Low Lethal Dose Research Articles

Quantification of saxitoxin in human blood by ELISA

Saxitoxin (STX) is a potent marine toxin that causes paralytic shellfish poisoning (PSP) which can result in significant morbidity and mortality in humans. Low lethal doses, rapid onset of PSP symptoms, and brief STX half-life in vivo require sensitive and rapid diagnostic techniques to monitor human exposures. Our laboratory has validated an enzyme-linked immunosorbent assay (ELISA) for quantitative detection of STX from 0.020 to 0.80 ng/mL in human whole blood and from 0.06 to 2.0 ng/mL in dried human blood which is simple, sensitive, rapid, and cost-effective. To our knowledge, this is the first validated method for the quantitation of saxitoxin in whole blood. Microsampling devices were used in sample collection which allows for standardized collection of blood, stable storage, and cost-efficient shipping. Quality control precision and accuracy were evaluated over the course of validation and were within 20% of theoretical concentrations. No detectable background concentrations of STX were found among fifty whole blood and dried blood convenience samples. Additionally, ten spiked individual whole blood and dried blood samples were tested for accuracy and precision and were within 20% of theoretical concentrations. Gonyautoxins 2&3 (GTX2&3) cross-reacted with this ELISA by 21%, but all other structurally related PSP toxins tested cross-reacted less than two percent. While clinical diagnosis or treatment of PSP would be unaffected by GTX2&3 cross-reactivity by ELISA, to accurately quantify individual PSP toxins, these results should be coupled with high performance liquid chromatography mass spectrometry measurements.

Two stable variants of Burkholderia pseudomallei strain MSHR5848 express broadly divergent in vitro phenotypes associated with their virulence differences.

Burkholderia pseudomallei (Bp), the agent of melioidosis, causes disease ranging from acute and rapidly fatal to protracted and chronic. Bp is highly infectious by aerosol, can cause severe disease with nonspecific symptoms, and is naturally resistant to multiple antibiotics. However, no vaccine exists. Unlike many Bp strains, which exhibit random variability in traits such as colony morphology, Bp strain MSHR5848 exhibited two distinct and relatively stable colony morphologies on sheep blood agar plates: a smooth, glossy, pale yellow colony and a flat, rough, white colony. Passage of the two variants, designated “Smooth” and “Rough”, under standard laboratory conditions produced cultures composed of > 99.9% of the single corresponding type; however, both could switch to the other type at different frequencies when incubated in certain nutritionally stringent or stressful growth conditions. These MSHR5848 derivatives were extensively characterized to identify variant-associated differences. Microscopic and colony morphology differences on six differential media were observed and only the Rough variant metabolized sugars in selective agar. Antimicrobial susceptibilities and lipopolysaccharide (LPS) features were characterized and phenotype microarray profiles revealed distinct metabolic and susceptibility disparities between the variants. Results using the phenotype microarray system narrowed the 1,920 substrates to a subset which differentiated the two variants. Smooth grew more rapidly in vitro than Rough, yet the latter exhibited a nearly 10-fold lower lethal dose for mice than Smooth. Finally, the Smooth variant was phagocytosed and replicated to a greater extent and was more cytotoxic than Rough in macrophages. In contrast, multiple locus sequence type (MLST) analysis, ribotyping, and whole genome sequence analysis demonstrated the variants’ genetic conservation; only a single consistent genetic difference between the two was identified for further study. These distinct differences shown by two variants of a Bp strain will be leveraged to better understand the mechanism of Bp phenotypic variability and to possibly identify in vitro markers of infection.

Lethal and sublethal effects of sulfoxaflor on the small brown planthopper Laodelphax striatellus

Laodelphax striatellus (Fallén), as an important pest of gramineous crops, has developed resistance to multiple classes of insecticides, impairing control efficiency. However, the application of insecticides is still the main control measure for it. Herein, the lethal effects of seven insecticides as well as sulfoxaflor, which has been introduced and registered for controlling L. striatellus in China, were investigated. The acute toxicity of these insecticides on the L. striatellus adults was ranked as sulfoxaflor > abamectin > dinotefuran > emamectin benzoate > ethofenprox > imidacloprid > chlorantraniliprole > chlorpyrifos. The toxicity of sulfoxaflor against the adults was highest with LD50 at 1.07–1.09ng/insect. In addition, the sublethal effects of lower lethal dose LD3 (0.06ng/insect), low lethal dose LD10 (0.15ng/insect) and moderate lethal dose LD30 (0.49ng/insect) of sulfoxaflor for L. striatellus were assessed as well. Both LD10 and LD30 induced slower nymphal development period, shorter oviposition period and longer pre-oviposition period in L. striatellus. The LD30 also shortened the longevity of females. Hormesis on fecundity was observed in L. striatellus exposed to LD3. Therefore, the net reproductive rate (R0) was increased by LD3. The intrinsic rate of increase (rm) was reduced by LD10 and LD30 while mean generation time (T) and doubling time (DT) were prolonged. The acute sulfoxaflor doses in its toxicological properties need to be considered when develop L. striatellus control strategy with sulfoxaflor. These results demonstrate that sulfoxaflor is a valid candidate for L. striatellus management.

Efficacy of diatomaceous earth and a DE-aerosol formulation against the common bed bug, Cimex lectularius Linnaeus in the laboratory

Wehaveinvestigatedefficacyofadiatomaceous earth(DE)andanaerosolformulationthereofforcontactand residual effects against the common bed bug, Cimex lectu- larius L. Efficacy of commercially available DE dust sam- ples varied with the product. DX13 TM and MotherEarth � dusts were significantly more effective than other DE dusts based on their lower lethal doses to kill 50 % of the insects, LD50 values (LD50s = 0.17 and 0.18 g m -2 , respectively), in contact bioassays with adult bugs. DX13 TM -aerosol demonstrated residual effect in both plastic box and the Petri dishmethods.Inlargeplasticboxesmortalityofthebedbugs due to residual effect of the aerosol was 98 % at 24 h. The residual effect of DX13 TM -aerosol in Petri dishes persisted for 21 days. Time to kill 50 % of the insects, LT50 value, increased from 10.2 h for freshly applied DE dust to 46.1 h fordustaged for21 days in Petri dishes. Mortalityof thebed bugsinthePetridisheswas(97 %at72 h,notonlyforfresh DX13 TM -aerosol residue, but also for residues aged up to 14 days. Mortality was 81 % at 72 h for DX13 TM -aerosol residueagedfor21 days.Mortalityofbedbugsconfinedona DX13 TM -incorporated mattress was 100 % at 48 and 72 h for bugs introduced on the mattress 3 and 32 weeks after manufacture, respectively. We also observed horizontal transfer of DX13 TM -dust between dead bugs collected from the DX13 TM -incorporated mattress to untreated ones. We discuss potential use of DX13 TM for the management of bed

Diversity and Toxicity of Bacillus thuringiensis from Shifting Cultivation (Jhum) Habitat.

Bacillus thuringiensis (Bt) strains were isolated from jhum-agriculture, jhum-forest, aquatic and fallow soil samples from Mizoram by acetate selection method. Isolates were characterized for biochemical typing, cry gene and protein profiling, growth curve study and toxicity against Culex tritaeniorhynchus. Bt frequency was high in jhum-agriculture land (69.56%) whereas low in jhum-forest soils (31.57%). Bt was found to be abundant in jhum shifting cultivation soil with an index ranging between 0.010 and 0.015. Majority of the isolates from jhum soils produced oval and spherical crystals and showed eleven types of crystal proteins groups. PCR analysis revealed predominance of dipteran-active cry genes (cry4 and cry9). The variations in crystal morphology, cry genes and Cry protein (s) from the isolates of Bt revealed molecular diversity. Higher mortality, lower lethal dose, and lesser time to kill were observed in Bt isolates from jhum soils than aquatic and fallow habitats. Based on the toxicity test, SC1 and HP7 isolates containing cry 4 and cry 9 genes showed higher activity. Growth curve analysis showed significant variations among Bt isolates to reach the sporulating stage. Higher growth index and lower mean generation time were observed in SC1 and HP7 Bt isolates. Bt strains express different endotoxin genes and crystal proteins and their harvesting time also varied from strain to strain. Significant variation was found in Bt isolates from jhum habitats in relation to the cry gene composition, protein profiling and toxicity. Results from this study suggest that novel Bt entomopathogens may complement for regulating mosquito vectors.

Assessment of Sublethal and Transgenerational Effects of Pirimicarb on the Wheat Aphids Rhopalosiphum padi and Sitobion avenae.

The wheat aphids, Rhopalosiphum padi (Linnaeus) and Sitobion avenae (Fabricius), are key pests on wheat crops worldwide. Management practices rely primarily on insecticides. The pirimicarb (carbamate) is used extensively as an effective insecticide to control these two aphids. In addition to the mortality caused by pirimicarb, various sublethal effects may occur in aphids when exposed to low lethal or sublethal doses. Understanding the general effect of pirimicarb on aphids could help increasing rational use of this insecticide. Under laboratory conditions, we assessed the sublethal effects of a low lethal concentration of pirimicarb (LC25) on biological traits and acetylcholinesterase (AChE) activity of R. padi and S. avenae. Both direct and transgenerational effects, i.e. on parent and the F1 generations were assessed, respectively. We found that R. padi and S. avenae responded differentially to the LC25 of pirimicarb. The parent generation of R. padi showed a 39% decrease in fecundity and multiple transgenerational effects were observed in the F1 generation; overall juvenile development, reproductive period, adult longevity and lifespan were longer than those of the control group. By contrast, LC25 of pirimicarb showed almost no effects on S. avenae biological traits in both the parent and F1 generations; only the pre-reproductive duration was reduced in F1 generations. Demographic parameter estimates (e.g. rm) showed similar trend, i.e. significant negative effect on R. padi population growth and no effect on S. avenae. However, AChE activity decreased in both R. padi and S. avenae treated by the LC25 of pirimicarb. We demonstrated sublethal and transgenerational effects of pirimicarb in the two wheat aphid species; it hinted at the importance of considering sublethal effects (including hormesis) of pirimicarb for optimizing Integrated Pest Management (IPM) of wheat aphids.

Lethal and sublethal effects of cycloxaprid, a novel cis-nitromethylene neonicotinoid insecticide, on the mirid bug Apolygus lucorum

Recently, Apolygus lucorum (Hemiptera: Miridae) has become an important insect pest of Bt cotton and fruit trees in China. At present, the application of chemical insecticides is the main control option for this pest. We investigated the lethal effect of seven neonicotinoid insecticides on A. lucorum adults, including six commercialized neonicotinoids and a novel one, cycloxaprid, which is developed in China. In addition, we assessed the sublethal physiological (longevity, fecundity) and behavioral (pre-oviposition and oviposition periods) effects of LD10 (low lethal dose) and LD40 (moderate lethal dose) of cycloxaprid on the mirid bug. Lethal effect of the insecticides was determined using a topical exposure method; contact toxicity against the adults was ranked as follows: cycloxaprid, nitenpyram > clothianidin > acetamiprid, imidacloprid > thiamethoxam > dinotefuran. Among these insecticides, the toxicity of cycloxaprid against the adults was the highest; its LD50 value was 2.54 ng a.i. (active ingredient)/adult. Both LD10 (0.74 ng a.i./adult) and LD40 (1.98 ng a.i./adult) of cycloxaprid induced sublethal effects in the adults; we observed a reduced longevity in males and the LD40 also shortened the longevity of females. In addition, the LD40 prolonged the pre-oviposition period and shortened the oviposition period of females, and it decreased their fecundity. The hatching rate of eggs laid by the females exposed to LD10 and LD40 greatly decreased (two-fold lower when exposed to LD40). Our results demonstrate that cycloxaprid is among the best candidate insecticides for controlling A. lucorum, and it showed significant lethal and sublethal effects on this mirid bug.

Bacterial overexpression of recombinant heteroscorpine-1 (rHS-1), a toxin from Heterometrus laoticus scorpion venom: trends for antibacterial application and antivenom production.

Heteroscorpine-1 (HS-1) was identified as a member of the scorpine family. HS-1 shows insecticidal activities, exhibiting a low median lethal dose (LD50) in mealworm (Tenebrio molitor L.) and inhibitory activities against Bacillus subtilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. In this study, a recombinant HS-1 (rHS-1) was produced by overexpression in E. coli. A large yield of product was obtained. The structure of purified rHS-1 was confirmed through mass spectrometry. Both anti-crude venom and anti-rHS-1 antibodies specifically recognized rHS-1, suggesting its structural similarity. Reactivated rHS-1 caused roughening and blebbing of bacterial cell surfaces. It showed higher activity than that of pre-refolded protein. Antisera raised against a partially purified and mis- or unfolded peptide can inhibit relevant bioactivity.

Association of the Outer Membrane Protein Omp33 With Fitness and Virulence of Acinetobacter baumannii

Outer membrane protein 33 (Omp33) is an outer membrane porin of Acinetobacter baumannii associated with carbapenem resistance. However, the role of Omp33 in the fitness and virulence of A. baumannii remains unknown. In the present study, we investigated the role of Omp33 in fitness and virulence of A. baumannii by using an isogenic knockout strain deficient in the omp33 gene (JPAB02), derived from the ATCC 17978 wild-type (wt). Both in vitro and in vivo defect in the growth rate was found in the JPAB02 strain in competition with the ATCC 17978 wt, highlighting the effect of Omp33 on the metabolic fitness. A significant reduction was observed both in adherence and invasion of human lung epithelial cells and in cytotoxicity of these cells and macrophages with JPAB02. In a murine peritoneal sepsis model, the JPAB02 strain exhibited lower lethal dose 0 (LD0), LD50, and LD100, and dissemination in mice, with reduced bacterial concentration in spleen and lungs. From these data, we concluded that Omp33 plays an important role for fitness and virulence of A. baumannii.

Efficacy of pesticides on the functional response on larval ectoparasitoid, Habrobracon hebetor Say (Hymenoptera: Braconidae)

Exposing to sub-lethal and low lethal doses of pesticides may cause changes in natural enemy behavioural, such as functional, response. In this study, the effects of chlorpyrifos, carbaryl, abamectin and spinosad were evaluated on the functional response of the Habrobracon hebetor to different densities of last instar larvae of Anagasta kuehniella Zeller. Young adult females of the parasitoid were exposed to LC30 of chlorpyrifos, carbaryl, abamectin and spinosad that were 0.32, 4.03, 3.05 and 17.51 mg a.i./l for 24 h, respectively. Host densities of 2, 4, 8, 16, 32 and 64 were offered to treated young females for 2 h in 10-cm Petri dishes and then the parasitism data were recorded. Experiments were conducted in eight replications. Functional response type was determined using logistic regression and the parameters were appraised by non-linear regression using statistical analysis software. Functional response was type Ш in control and insecticide treatments. Searching efficiency in control, chlorpyrifos-, carbaryl-, abamectin- and spinosad-treated wasps were 0.008 ± 0.002, 0.002 ± 0.0009, 0.0034 ± 0.0013, 0.0076 ± 0.002 and 0.0073 ± 0.002 h−1and handling times were 1.38 ± 0.1, 7.64 ± 1.01, 3.3 ± 0.315, 1.55 ± 0.1 and 1.46 ± 0.11 h, respectively. Chlorpyrifos and carbaryl had the highest effect on searching efficiency of H. hebetor. Spinosad and abamectin showed less adverse effect on the functional response parameters. Finally, after conducting the advanced field studies, spinosad and abamectin may be used as a compatible chemical material with biological control agent in integrated pest management programmes.

Specific antibodies protect gamma-irradiated mice against Francisella tularensis infection

The role of antibodies in the course of Francisella tularensis (F. tularensis) infection is still a subject of debate. The understanding of the poorly described role of humoral immunity is more than important for the effort to develop effective prophylactic procedure against the infection with Francisella virulent strains. We utilized the model of gamma-irradiated mice for the studies of the protective role of anti-F. tularensis antibodies in order to partially eliminate cellular responses. The model of gamma-irradiated mice can also demonstrate the responses of immunocompromised host to intracellular bacterial infection. The gamma-irradiation by doses greater than 3 Gy completely impairs the resistance to infection and causes a disbalance of cytokine production in mice. In this study, we demonstrate that passive transfer of immune sera protected irradiated mice against subsequent infection with strains of F. tularensis subsp. holarctica. Naïve mice of BALB/c or C3H/CBi strains were subjected to passive transfer of sera obtained from immunized mice with live vaccine strain (LVS) F. tularensis LVS, F. tularensis subsp. holarctica strain 15, heat-killed F. tularensis LVS, or heat-killed strain 15 two hours before infection with lethal doses of LVS or strain 15. The passive transfer of sera obtained from immunized mice conferred full protection of naïve unirradiated as well as sublethally irradiated mice against low lethal doses of infection with F. tularensis LVS or strain 15, in all variants of the experiments. In addition, the passively protected mice that survived the primary infection with F. tularensis LVS were protected also against further secondary challenge with a highly virulent strain of F. tularensis subsp. tularensis SchuS4. Moreover, the first evidence of combination of successful passive transfer of immunity by specific antisera and subsequent active immunization of immunocompromised animals is demonstrated. In summary, we demonstrate that B cell-mediated effector responses together with the induction of T cell-mediated immunity both play an important role in naïve and also in immunocompromised mice and this fact it would be appropriate to take into the account in the design of new vaccines.

Snail abundance in freshwater canals in the eastern province of Saudi Arabia and acute toxicity studies of copper sulphate in Biomphalaria arabica and Lymnaea auricularia

The present study aimed to investigate the distribution of three species of freshwater snails, namely; Biomphalaria Arabica ; Planorbidae, Lymnaea auricularia ; Lymnaeidae and Melanoides tuberculatus ; Thiaridae in two different parallel canals (the concrete irrigation and the earthen drainage canals) in the eastern province of Saudi Arabia and the effect of copper sulphate pentahydrate (CuSO 4 .5H 2 O) on mortality of the first two species after 24 h of exposure. The physico-chemical properties of the water especially, temperature and pH were determined as important factors for snail distribution. The results show that the water temperature and the pH of the two canals were almost the same. The range of water temperature was 25.8 to 28.3°C, while the water pH was 6.6 to 6.8. The two canals were occupied by three species of aquatic snails viz. B. arabica , L. auricularia and M. tuberculatus . The bioassay of CuSO 4 .5H 2 O on only two snail species showed 24 h–LC 50 as 5.7 ppm for B. arabicus and 7.8 ppm for L. auricularia. This result reveals that CuSO 4 .5H 2 O was highly effective in snail eradication. On the basis of the present study, it is recommended that some further studies on the canals including all environmental factors should be investigated for their role in aquatic snail distribution. However, the chronic effect of lower lethal dose of CuSO 4 .5H 2 O on the snails mortality and their effects on developmental stages should be investigated, and some local plant extract should also be tried for their potency on snail mortality. Key words : Biomphalaria, Lymnea, Melanoides , distribution, freshwater snails, copper sulphate, Saudi Arabia.

Assessment of physiological sublethal effects of imidacloprid on the mirid bug Apolygus lucorum (Meyer-Dür)

Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae) is currently one of major mirid pests in the Yangtze River and the Yellow River regions in China. Imidacloprid (neonicotinoid) is widely used against pierce-sucking pest insects, including against A. lucorum. In addition to its direct lethal effect, multiple negative sublethal effects may also occur in exposed insects. We assessed potential sublethal effects of imidacloprid on some biological characteristics of A. lucorum with the aim of increasing rational use of imidacloprid against that cotton pest. The lethal toxicity of imidacloprid on adults of A. lucorum was determined in laboratory conditions by a topical application bioassay (LD(50) = 6.70 ng a.i. [active ingredient]/A. lucorum adult). We also estimated a sublethal dose, LD(5) (0.38 ng a.i./adult), a low lethal dose, LD(25) (1.96 ng a.i./adult), and moderate lethal dose, LD(40) (3.97 ng a.i./adult). The sublethal dose of imidacloprid (LD(5)) shortened the pre-oviposition period of females but increased the time required for eggs to develop (i.e. longer embryogenesis). The low lethal dose (LD(25)) also reduced the pre-oviposition period. Females exposed to the LD(40) laid eggs that developed faster but overall percentage of eggs hatching was reduced. LD(25) and LD(40) reduced longevity of males but not of females. In addition, the susceptibility to seven insecticides generally used on Chinese crops was not modified in A. lucorum previously exposed to the LD(25) of imidacloprid. Our results demonstrate sublethal effects of low doses of imidacloprid on A. lucorum (notably on pre-oviposition period and egg development) which may have an impact on population dynamics of this pest.

Searching for new chemotherapies for tropical diseases: ruthenium-clotrimazole complexes display high in vitro activity against Leishmania major and Trypanosoma cruzi and low toxicity toward normal mammalian cells.

Eight new ruthenium complexes of clotrimazole (CTZ) with high antiparasitic activity have been synthesized, cis,fac-[Ru(II)Cl(2)(DMSO)(3)(CTZ)] (1), cis,cis,trans-[Ru(II)Cl(2)(DMSO)(2)(CTZ)(2)] (2), Na[Ru(III)Cl(4)(DMSO)(CTZ)] (3), Na[trans-Ru(III)Cl(4)(CTZ)(2)] (4), [Ru(II)(η(6)-p-cymene)Cl(2)(CTZ)] (5), [Ru(II)(η(6)-p-cymene)(bipy)(CTZ)][BF(4)](2) (6), [Ru(II)(η(6)-p-cymene)(en)(CTZ)][BF(4)](2) (7), and [Ru(II)(η(6)-p-cymene)(acac)(CTZ)][BF(4)] (8) (bipy = bipyridine; en = ethlylenediamine; acac = acetylacetonate). The crystal structures of compounds 4-8 are described. Complexes 1-8 are active against promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi. Most notably, complex 5 increases the activity of CTZ by factors of 110 and 58 against L. major and T. cruzi, with no appreciable toxicity to human osteoblasts, resulting in nanomolar and low micromolar lethal doses and therapeutic indexes of 500 and 75, respectively. In a high-content imaging assay on L. major-infected intraperitoneal mice macrophages, complex 5 showed significant inhibition on the proliferation of intracellular amastigotes (IC(70) = 29 nM), while complex 8 displayed some effect at a higher concentration (IC(40) = 1 μM).

Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis

BackgroundBurkholderia pseudomallei is the causative agent of melioidosis, a tropical disease of humans with a variable and often fatal outcome. In murine models of infection, different strains exhibit varying degrees of virulence. In contrast, two related species, B. thailandensis and B. oklahomensis, are highly attenuated in mice. Our aim was to determine whether virulence in mice is reflected in macrophage or wax moth larvae (Galleria mellonella) infection models.ResultsB. pseudomallei strains 576 and K96243, which have low median lethal dose (MLD) values in mice, were able to replicate and induce cellular damage in macrophages and caused rapid death of G. mellonella. In contrast, B. pseudomallei strain 708a, which is attenuated in mice, showed reduced replication in macrophages, negligible cellular damage and was avirulent in G. mellonella larvae. B. thailandensis isolates were less virulent than B. pseudomallei in all of the models tested. However, we did record strain dependent differences. B. oklahomensis isolates were the least virulent isolates. They showed minimal ability to replicate in macrophages, were unable to evoke actin-based motility or to form multinucleated giant cells and were markedly attenuated in G. mellonella compared to B. thailandensis.ConclusionsWe have shown that the alternative infection models tested here, namely macrophages and Galleria mellonella, are able to distinguish between strains of B. pseudomallei, B. thailandensis and B. oklahomensis and that these differences reflect the observed virulence in murine infection models. Our results indicate that B. oklahomensis is the least pathogenic of the species investigated. They also show a correlation between isolates of B. thailandensis associated with human infection and virulence in macrophage and Galleria infection models.

Enhanced immunogenicity of a novel Stx2Am-Stx1B fusion protein in a mice model of enterohemorrhagic Escherichia coli O157:H7 infection

Shiga toxins (Stxs) which include Stx1 and Stx2 produced by EHEC O157:H7 are responsible for severe diseases, including hemolytic uremic syndrome (HUS) in humans. In our previous study, a fusion protein Stx2B-Stx1B (2S for short) was prepared and displayed immunogenicity against low lethal dose challenge of E. coli O157:H7. To enhance the immunogenicity against both toxins above, we constructed a novel fusion protein carrying the Stx1B subunit and enzyme-inactive Stx2A subunit, designated Stx2Am-Stx1B (SAmB for short). The fusion protein SAmB elicited high level humoral IgG and IgG1 in mice and induced Th2-typical cytokines IL-4/IL-10 but not Th1-typical cytokine INF-γ, indicating a partial to humoral immunoresponse mediated by Th2-type cells that contributed to this humoral reactivity. Higher level neutralizing antibodies against Stx2 were elicited by SAmB than 2S. An enhanced effect of protection (93.3%) against high lethal dose challenge of lysed E. coli O157:H7 was observed, and the SAmB also provided cross protection against purified Stx1 and Stx2.

Different Routes of Inoculation Impact Infectivity and Pathogenesis of H5N1 High Pathogenicity Avian Influenza Virus Infection in Chickens and Domestic Ducks

The H5N1 type A influenza viruses classified as Qinghai-like virus (clade 2.2) are a unique lineage of type A influenza viruses with the capacity to produce significant disease and mortality in gallinaceous and anseriform birds, including domestic and wild ducks. The objective of this study was to determine the susceptibility and pathogenesis of chickens and domestic ducks to A/Whooper Swan/Mongolia/224/05 (H5N1) high pathogenicity avian influenza (HPAI) virus when administered through respiratory or alimentary routes of exposure. The chickens and ducks were more susceptible to the H5N1 HPAI virus, as evidenced by low infectious and lethal viral doses, when exposed by intranasal as compared to alimentary routes of inoculation (intragastric or oral-fed infected chicken meat). In the alimentary exposure pathogenesis study, pathologic changes included hemorrhage, necrosis, and inflammation in association with virus detection. These changes were generally observed in most of the visceral organs of chickens, between 2 and 4 days postinoculation (DPI), and are similar to lesions and virus localization seen in birds in natural cases or in experimental studies using the intranasal route. Alimentary exposure to the virus caused systemic infection in the ducks, characterized by moderate lymphocytic encephalitis, necrotized hepatitis, and pancreatitis with a corresponding demonstration of virus within the lesions. In both chickens and ducks with alimentary exposure, lesions, virus, or both were first demonstrated in the upper alimentary tract on 1 DPI, suggesting that the alimentary tract was the initial site affected upon consumption of infected meat or on gavage of virus in liquid medium. However, as demonstrated in the infectivity study in chickens, alimentary infection required higher exposure doses to produce infection as compared to intranasal exposure in chickens. These data suggest that upper respiratory exposure to H5N1 HPAI virus in birds is more likely to result in virus infection and transmission than will consumption of infected meat, unless the latter contains high doses of virus, as found in cannibalized infected carcasses.

Evaluation on Cytotoxicity and Genotoxicity of the Exfoliated Silicate Nanoclay

The concern about toxicity for nanosilicate platelets (NSP) derived from natural montmorillonite clay is addressed. The NSP nanoclay was isolated from polyamine-salt exfoliation of the layered silicate clay into randomized individual plates, possessing multiple ionic charges on the surface of silicate plates with an average geometric dimension of ca. 80 x 80 x 1 nm(3). The material had been previously shown to be effective for antimicrobial and tendency for adhering onto the biomaterial surface based on the direct observation by using scanning electron microscope. The material safety on genotoxic effect was investigated by using three different test systems: the Comet assay test on Chinese Hamster Ovary (CHO) cells in vitro, micronucleus (MN) assay in vivo and the Salmonella gene mutation assay on strain TA98, TA100, TA102, TA1535 and TA1537. The Comet assay showed no DNA damage after 24 h of incubation with NSP of 1000 microg/mL. The MN test indicated no significant micronucleus induction in the CHO cells at the concentrations tested. With all five strains of Salmonella typhimurium, none of mutations was found. Furthermore, cytotoxicity of the same material was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release, showing a low cytotoxicity on CHO cells below 1000 microg/mL after 12 h incubation period and a dose-dependent effect after 24 h incubation. For feeding to rats, the acute oral toxicity was shown a low lethal dose (LD(50)) or greater than 5700 mg/kg body weight for both male and female Sprague-Dawley rats. Overall, the study has demonstrated the safety of the NSP for potential uses in biomedical areas.

Infectious and Lethal Doses of H5N1 Highly Pathogenic Avian Influenza Virus for House Sparrows (Passer Domesticus) and Rock Pigeons (Columbia Livia)

Terrestrial wild birds commonly associated with poultry farms have the potential to contribute to the spread of H5N1 highly pathogenic avian influenza (HPAI) virus within or between poultry facilities or between domesticated and wild bird populations. This potential, however, varies between species and is dependent on several virus and host factors, including habitat utilization, susceptibility, and viral shedding patterns. To provide data on susceptibility and shedding patterns of house sparrows (Passer domesticus) and rock pigeons (Columba livia), 20 birds from each species were inoculated with decreasing concentrations of A/whooper swan/Mongolia/244/05 (H5N1) HPAI virus, and the birds were evaluated for morbidity, mortality, viral shedding, and seroconversion over a 14-day trial. The house sparrows were highly susceptible to the H5N1 HPAI virus as evidenced by low infectious and lethal viral doses. In addition, house sparrows excreted virus via the oropharynx and cloaca for several days prior to the onset of clinical signs. Based on these results, house sparrows could play a role in the dissemination of H5N1 HPAI virus in poultry. In contrast, pigeons were resistant to the HPAI virus, requiring a high concentration of virus to produce infection or death. When infection did occur, the duration of viral shedding was brief, and viral titers were low. The data suggests that pigeons would contribute little to the transmission and spread of H5N1 HPAI virus in poultry.

The radiobiological effectiveness of carbon-ion beams on growing neurons

Purpose: Recently carbon-ion beams have been reported to be remarkably effective for controlling various cancers with less toxicity and are thought to be a promising modality for cancer treatment. However, the biological effect of carbon-ion beams arising on normal neuron remains unknown. Therefore, this study was undertaken to investigate the effect of carbon-ion beams on neurons by using both morphological and functional assays.Materials and methods: Dorsal root ganglia (DRG) and sympathetic ganglion chains (SYMP) were isolated from day-8 and day-16 chick embryos and cultured for 20 h. Cultured neurons were exposed to carbon-ion beams and X-rays. Morphological changes, apoptosis and cell viability were evaluated with the Growth Cone Collapse (GCC), Terminal deoxynucleotidyl Transferase (TdT)-mediated deoxyUridine TriPhosphate (dUTP) nick End Labeling [TUNEL] assay and 4-[3-(4-iodophenyl)- 2-(4-nitrophenyl)- 2H-5-tetrazolio]- 1,3-benzenedisulfonate [WST-1] assays, respectively.Results: Irradiation caused GCC and neurite destruction on a time- and irradiation dose-dependent manner. Changes in morphological characteristics were similar following either irradiation. Morphological and functional assays showed that day-8 neurons were more radiosensitive than day-16 neurons, whereas, radiosensitivity of DRG was comparable to that of SYMP. The dose-response fitting curve utilising both GCC and TUNEL labeling index showed higher relative biological effectiveness (RBE) values were associated with lower lethal dose (LD) values, while lower RBE was associated with higher LD values.Conclusion: Exposure to high-linear energy transfer (LET) irradiation is up to 3.2 more efficient to induce GCC and apoptosis, in early developed neuronal cells, than low-LET irradiation. GCC is a reliable method to assess the radiobiological response of neurons.