Comparative Evaluation of Anthelmintic Activity of Edible and Ornamental Pomegranate Ethanolic Extracts against Schistosoma mansoni.

Due to the development of praziquantel (PZQ) schistosomes resistant strains, the discovery of new antischistosomal agents is of high priority in research. This work reported the in vitro and in vivo effects of the edible and ornamental pomegranate extracts against Schistosoma mansoni. Leaves and stem bark ethanolic extracts of both dried pomegranates were prepared at 100, 300, and 500 µg/mL for in vitro and 600 and 800 mg/kg for in vivo. Adult worms Schistosoma mansoni in RPMI-1640 medium for in vitro and S. mansoni infected mice for in vivo tests were obtained from Theodor Bilharz Research Institute, Cairo, Egypt. In vitro activity was manifested by significant coupled worms separation, reduction of motor activity, lethality, and ultrastructural tegumental alterations in adult worms. In vivo activity was manifested revealed by significant reduction of hepatic granulomas number and diameter, decreased number of bilharzial eggs in liver tissues, lowered liver inflammatory infiltration, decreased hepatic fibrosis, and inducible nitric oxide synthase (iNOS) expression. Ethanolic stem bark extract of edible pomegranate exhibited highest antischistosomal activities both in vitro and in vivo. Therefore, pomegranate showed a good potential to be used as a promising new candidate for the development of new schistosomicidal agents.

Independent action between DvSnf7 RNA and Cry3Bb1 protein in southern corn rootworm, Diabrotica undecimpunctata howardi and Colorado potato beetle, Leptinotarsa decemlineata.

In recent years, corn rootworm (CRW)-resistant maize events producing two or more CRW-active Bt proteins have been commercialized to enhance efficacy against the target pest(s) by providing multiple modes of action (MoA). The maize hybrid MON 87411 has been developed that produces the CRW-active Cry3Bb1 Bt protein (hereafter Cry3Bb1) and expresses a RNAi-mediated MoA that also targets CRW. As part of an environmental risk assessment for MON 87411, the potential for an interaction between the CRW-active DvSnf7 RNA (hereafter DvSnf7) and Cry3Bb1 was assessed in 12-day diet incorporation bioassays with the southern corn rootworm (SCR, Diabrotica undecimpunctata howardi). The potential for an interaction between DvSnf7 and Cry3Bb1 was evaluated with two established experimental approaches. The first approach evaluated each substance alone and in combination over three different response levels. For all three response levels, observed responses were shown to be additive and not significantly different from predicted responses under the assumption of independent action. The second approach evaluated the potential for a fixed sub-lethal concentration of Cry3Bb1 to decrease the median lethal concentration (LC50) of DvSnf7 and vice-versa. With this approach, the LC50 value of DvSnf7 was not altered by a sub-lethal concentration of Cry3Bb1 and vice-versa. In addition, the potential for an interaction between the Cry3Bb1 and DvSnf7 was tested with Colorado potato beetle (CPB, Leptinotarsa decemlineata), which is sensitive to Cry3Bb1 but not DvSnf7. CPB assays also demonstrated that DvSnf7 does not alter the activity of Cry3Bb1. The results from this study provide multiple lines of evidence that DvSnf7 and Cry3Bb1 produced in MON 87411 have independent action.

Zingerone suppresses liver inflammation induced by antibiotic mediated endotoxemia through down regulating hepatic mRNA expression of inflammatory markers in Pseudomonas aeruginosa peritonitis mouse model.

Antibiotic-induced endotoxin release is associated with high mortality rate even when appropriate antibiotics are used for the treatment of severe infections in intensive care units. Since liver is involved in systemic clearance and detoxification of endotoxin hence it becomes a primary target organ for endotoxin mediated inflammation. Currently available anti-inflammatory drugs give rise to serious side effects. Hence, there is an urgent need for safe and effective anti-inflammatory therapy. It is likely that anti-inflammatory phytochemicals and neutraceutical agents may have the potential to reduce the endotoxin mediated inflammation and complications associated with endotoxin release. Keeping this in mind, the present study was planned to evaluate the hepatoprotective potential of zingerone (active compound of zingiber officinale) against liver inflammation induced by antibiotic mediated endotoxemia. The selected antibiotics capable of releasing high content of endotoxin were employed for their in vivo efficacy in P.aeruginosa peritonitis model. Released endotoxin induced inflammation and zingerone as co-anti-inflammatory therapy significantly reduced inflammatory response. Improved liver histology and reduced inflammatory markers MDA, RNI, MPO, tissue damage markers (AST, ALT, ALP) and inflammatory cytokines (MIP-2, IL-6 and TNF-α) were indicative of therapeutic potential of zingerone. The mechanism of action of zingerone may be related to significant inhibition of the mRNA expression of inflammatory markers (TLR4, RelA, NF-kB2, TNF- α, iNOS, COX-2) indicating that zingerone interferes with cell signalling pathway and suppresses hyper expression of cell signaling molecules of inflammatory pathway. Zingerone therapy significantly protected liver from endotoxin induced inflammatory damage by down regulating biochemical as well as molecular markers of inflammation. In conclusion, this study provides evidence that zingerone is a potent anti-inflammatory phytomedicine against hepatic inflammation induced by antibiotic mediated endotoxemia. These results thus suggest that zingerone treatment can be used as a co-therapy with antibiotics to reduced endotoxin induced inflammation during treatment of severe P.aeruginosa infections.

Potato flour mediated solid-state fermentation for the enhanced production of Bacillus thuringiensis-toxin.

In this study, we explored the efficacy of raw potato flour (PF) as supplement to the conventional LB medium (LB control, designated as M1) for enhancing the concomitant production of endospores and δ-endotoxin from Bacillus thuringiensis subsp. kurstaki by solid-state fermentation (SSF). Of different concentrations and combinations of media tested, 10% (w/v) PF supplemented LB medium (M2) was found as the best source for the maximum yield of toxin. After 12 h submerged fermentation (SmF) at 37°C and 125 rpm, M2 was made into a wet-solid matter for SSF by removing the supernatant (1000 ×g, 10 min); the resultant pellet subsequently incubated statically (37°C) for the production of B. thuringiensis subsp. kurstaki toxin (Btk-toxin). In comparison to M1, yield of δ-endotoxin purified by sucrose density gradient centrifugation method from M2 was about 6-fold higher (53% recovery). This maximum yield from M2 was obtained at 48 h (as against 72 h from M1), thus the gestation period of M2 was reduced by 24 h with higher yield. In addition to the quantitative data, qualitative photomicrographs taken by image analyzer, scanning electron and fluorescent microscopes and digital camera showed physical evidences for the upper hand of SSF over conventional SmF for the enhanced production of Btk-toxin. SDS-PAGE image of the purified δ-endotoxin showed three major fractions with apparent MWs 66, 45 and 30 kDa. Briefly, if low-cost agricultural products like PF is used as supplement to LB, by SSF strategy, production of Btk-toxin could be enhanced to 6-fold in short gestation time without losing its entomotoxicity efficiency.

The residual occurrences of Bacillus thuringiensis biopesticides in food and beverages.

In 2006, 54 pasteurized full fat milk samples, 40 ice-cream samples, and two green-tea beverage samples were analyzed and a total of 19 Bacillus thuringiensis-like strains were isolated, nine from seven pasteurized milks, one from an ice-cream with peach pulp and juice, and nine from two green-tea beverages. These strains were classified as B. thuringiensis, contained the cry1A gene and produced crystal inclusions during sporulation. All strains were characterized by a serotyping test, SDS-PAGE, random amplified polymorphic DNA, and enterotoxic gene PCR analysis. Most isolates produced bipyramidal crystals and belonged to serotypes H3a3b, H5a5b, or H7. Furthermore, two strains from pasteurized full fat milks and three strains from green-tea beverages were indistinguishable from the B. thuringiensis subsp. kurstaki strains isolated from commercial biopesticides (Kaiyan, Qiangdi, Lvpuan and Sutai), suggesting the residual occurrences of B. thuringiensis from biopesticides in food and beverages.

Bitrophic and tritrophic effects of Bt Cry3A transgenic potato on beneficial, non-target, beetles.

Insect-resistant transgenic plants have been suggested to have unpredictable effects on the biodiversity of the agro-ecosystem, including potential effects on insect natural enemies, beneficial in control of crop pests. Whilst carnivorous as adults, many of these predators may also consume plant tissues, in particular plant pollen and nectar. Coleoptera are important in terms of agro-ecological research not only because of the large number of species in this order, but also because of their role as biological control agents. Thus any detrimental impact on this group of insects would be highly undesirable. The effects of potato expressing the coleopteran-specific Bacillus thuringiensis delta-endotoxin Cry3A (Bt Cry3A) on the ladybird beetle Harmonia axyridis and the carabid beetle Nebria brevicollis were investigated via the bitrophic interaction of the adult ladybird with potato flowers and the tritrophic interaction of the carabid consuming a non-target potato pest. Immunoassays confirmed accumulation of the transgene product in potato leaves and floral tissues (at levels of up to 0.01% (pollen) and 0.0285% (anthers) of total soluble protein). Despite H. axyridis and N. brevicollis belonging to the targeted insect order, no significant effects upon survival or overall body mass change of either beetle were observed. Furthermore, Bt Cry3A had no detrimental effects on reproductive fitness of either beetle species, either in terms of fecundity or subsequent egg viability. Behavioural analysis revealed no significant impact of Bt Cry3A on beetle activity or locomoter behaviour. Ligand blots indicate that this is due to either the absence of Bt-binding sites in brush border membrane vesicles (BBMV) isolated from Nebria brevicollis, or in the case of Harmonia axyridis, the binding did not functionally lead to behavioural or physical effects.

Influence of media composition on the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis.

Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of delta-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/liter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against fifth-instar Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x 10(6)/mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x 10(6)/mg, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced delta-endotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard.

[Influence of various ions in sporulation and the formation of delta-endotoxin in Bacillus thuringiensis cultures]. / Influencia de algunos iones en la esporulación y formación de delta-endotoxina en cultivos de Bacillus thuringiensis.

This paper deals with studies related to the influence of several ions on growth, spore formation and endotoxin formation by a Bacillus thuringiensis HD-1 strain commonly used for bioinsecticide production. Two basal media (4 and 5, Table 1) containing glucose, (NH4)2 SO4, phosphates and yeast extract or bacto peptone as organic nitrogen sources were supplemented with several ions in different concentrations, as shown in table 1. The experiments were conducted in 1000 ml Erlenmeyer flasks, containing 100 ml of medium, located in a rotary shaker at 30 degrees C. Several estimations were carried out, mainly biomass by optical density and colony forming units (CFU), glucose, Mg+2 and Mn+2 consumption and delta-endotoxin by a rocket immunoelectrophoretic method. The results obtained (Table 2) clearly show the importance of the addition of Ca+2, Mg+2 and Mn+2 to the basal media, because the highest values of CFU/l and delta-endotoxin (expressed as protein in g/l) were achieved in those media supplemented with the ions mentioned. It was also proved that the supplementation with Ca+2 was also essential for maintaining the thermal stability of the spores (Table 3). It can be concluded that an adequate formulation of media mainly related with the content of Mn+2, Mg+2 and Ca+2 is essential for obtaining high yields of spore crystal production of a Bacillus thuringiensis HD-1 strain.