Larvicidal and adulticidal effects and ultrastructural changes of larvae midgut epithelium of musca domestica (diptera: muscidae) fed with bacillus thuringiensis var. kyushuensis

Abstract INTRODUCTION: Musca domestica is resistant to many insecticides; hence, biological control is a suitable alternative. METHODS: We evaluated the lethality of strain Btk176 towards the larval and adult M. domestica and the histopathological effects in the larvae midgut. RESULTS: We observed 99% larval and 78.9% adult mortality within 48 hours of spore ingestion (dosage, 2.4×108 CFU/ml). The histopathological effects were consistent with cytotoxicity. PCR analysis showed the presence of the cry1Ba gene. Transmission electron microscopy revealed a bipyramidal parasporal body. Thurigiensin activity was not detected. CONCLUSIONS: The serovar, Btk176 might be a potential biocontrol agent for houseflies.

Selection of phytotoxin producing rhizobacteria

In order to select phytotoxin producing rhizobacteria to control weed plants, twenty five bacterial strains previously isolated from the rhizospheres of various plants were grown in a liquid medium and, after cell removal by centrifugation, the liquid phases were freeze-dried and the products were extracted with ethyl acetate/methanol. The extracts were concentrated to dryness under vacuum and dissolved in water and sucrose solution to be submitted to in vitro assays of lettuce (Lactuca sativa L.) seed germination and wheat (Triticum aestivum L.) coleoptile growth. Although most samples affected coleoptile growth, only those from four strains reduced lettuce seed germination. Two strains of Bacillus cereus, one strain of B. pumilus and one of Stenotrophoonas altophilia were the most promising microorganisms for producing phytotoxin and, consequently, for the development of new weed control products.

Com o objetivo de selecionar rizobactérias produtoras de fitotoxinas para uso no controle de plantas daninhas, vinte e cinco isolados bacterianos previamente obtidos das rizosferas de diferentes plantas foram cultivados em meio líquido e, após remoção das células por centrifugação, as fases líquidas foram liofilizadas e os resíduos obtidos foram submetidos à extração com acetato de etila/metanol. Os extratos foram concentrados sob vácuo até secura e dissolvidos em água e solução de sacarose para serem submetidos a testes in vitro de germinação de sementes de alface (Lactuca sativa L.) e de crescimento de coleóptilos de trigo (Triticum aestivum L.). Embora a maior parte das amostras tenha desfavorecido o crescimento dos coleóptilos de trigo, somente as provenientes de quatro isolados reduziram a germinação das sementes de alface. Dois isolados de Bacillus cereus, um isolado de B. pumilus e um de Stenotrophomonas maltophilia foram os microrganismos mais promissores para a produção de fitotoxinas, com possibilidade de uso no desenvolvimento de novos produtos para o controle de plantas daninhas.

Pseudomonas aeruginosa Exotoxin A Reduces Chemoresistance of Oral Squamous Carcinoma Cell via Inhibition of Heat Shock Proteins 70 (HSP70)

PURPOSE: Oral squamous carcinoma (OSCC) cells exhibit resistance to chemotherapeutic agent-mediated apoptosis in the late stage of malignancy. Increased levels of heat shock proteins 70 (HSP70) in cancer cells are known to confer resistance to apoptosis. Since recent advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers, we investigated the effect of Pseudomonas aeruginosa exotoxin A (PEA) on HSP70 expression and induction of apoptosis in chemoresistant OSCC cell line (YD-9). MATERIALS AND METHODS: The apoptotic effect of PEA on chemoresistant YD-9 cells was confirmed by MTT, Hoechst and TUNEL stains, DNA electrophoresis, and Western blot analysis. RESULTS: While YD-9 cells showed high resistance to chemotherapeutic agents such as etoposide and 5-fluorouraci (5-FU), HSP70 antisense oligonucelotides sensitized chemoresistant YD-9 cells to etoposide and 5-FU. On the other hand, PEA significantly decreased the viability of YD-9 cells by deteriorating the HSP70-relating protecting system through inhibition of HSP70 expression and inducing apoptosis in YD-9 cells. Apoptotic manifestations were evidenced by changes in nuclear morphology, generation of DNA fragmentation, and activation of caspases. While p53, p21, and E2F-1 were upregulated, cdk2 and cyclin B were downregulated by PEA treatment, suggesting that PEA caused cell cycle arrest at the G2/M checkpoint. CONCLUSION: Therefore, these results indicate that PEA reduced the chemoresistance through inhibition of HSP70 expression and also induced apoptosis in chemoresistant YD-9 cells.

Toxinas de Clostridium perfringens / Toxins of Clostridium perfringens

Clostridium perfringens es un bacilo grampositivo anaerobio con capacidad de formar esporas. Es uno de los patógenos bacterianos con mayor distribución en el medio ambiente, ya que puede ser aislado de muestras de suelo y de agua y además forma parte de la microbiota intestinal de animales y humanos. Sin embargo, en ciertas ocasiones puede actuar como patógeno oportunista y causar enfermedades como la gangrena gaseosa, la enterotoxemia del ovino y del caprino y la disentería del cordero, entre otras. En humanos, está asociado a enfermedades como la intoxicación por alimentos, la enterocolitis necrotizante en niños y la enteritis necrótica o pigbel de las tribus de Papúa-Nueva Guinea. El renovado interés que existe actualmente en el estudio de C. perfringens como patógeno veterinario y humano, junto con el avance de la biología molecular, han hecho posible que la ciencia tenga hoy un conocimiento más profundo sobre la biología y la patogenia de esta bacteria. En esta revisión bibliográfica se discuten y actualizan los principales aspectos de la patogenia intestinal de C. perfringens teniendo en cuenta las toxinas con mayor importancia médica descritas hasta el presente.

Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present.

Biolarvicides in vector control: challenges and prospects.

Biolarvicides, based on mosquitocidal toxins of certain strains of Bacillus sphaericus and Bacillus thuringiensis var israelensis H-14 (Bti) are highly effective against mosquito larvae at very low doses and safe to other non-target organisms. During past two decades various biolarvicide formulations produced in India and abroad have been tested at Malaria Research Centre and some formulations have undergone large-scale operational trials. Biolarvicide formulations of B. sphaericus are useful in the control of Culex and certain Anopheles spp, such as An. stephensi and An. subpictus, but not much effective against An. culicifacies and almost ineffective against Aedes aegypti. Repeated application of B. sphaericus in the same habitat, however, results in the development of resistance in larvae of target mosquitoes. In view of its low specificity for An. culicifacies and the potential for resistance in An. stephensi, B. sphaericus has limited prospects for control of malaria vectors. However, with some resistance management, B. sphaericus can still be used against Culex mosquitoes. On the other hand Bti formulations, which have broader spectrum of activity against Aedes, Culex and Anopheles spp, have not shown significant development of resistance in mosquitoes but their activity in field, particularly against surface feeding anopheline larvae is affected by various bioenvironmental factors, thus requiring weekly application in most habitats. To overcome this problem development of slow release formulations and genetically engineered biolarvicides by transplanting mosquitocidal toxin genes of Bti and B. sphaericus in some other environmentally compatible organisms have been investigated by different scientists.

Cross-resistance to Bacillus sphaericus strains in Culex quinquefasciatus resistant to B. sphaericus 1593M.

Bacillus sphaericus 1593M resistant larvae of Culex quinquefasciatus were reared in the laboratory since 1995. Resistance in the larvae was monitored by subjecting selection pressure using B. sphaericus 1593M at every generation. Bioassays were conducted with different strains of B. sphaericus (Bs 2297, Bs 2362 and Bs IAB 59) and confirmed cross-resistance in the present study. The level ranged between 27.3 to 18.2 fold in comparison with susceptible larvae. But Bacillus thuringiensis var israelensis strains (Bti PG14 and Bti 426) did not show any cross-resistance in the larvae and it emphasized a need to study the mode of action of B. sphaericus toxin that induces cross-resistance in the larval strain.

Effect of insecticidal crystal proteins of Bacillus thuringiensis var. israelensis on the enzymes of rat intestinal brush border membrane vesicles.

In vivo treatment of intestinal brush border membrane vesicles with solubilized insecticidal crystal proteins (ICP) from the two strains of B. thuringiensis var. israelensis (VCRC B17 and VCRC MB24) caused no adverse effect on gamma glutamyl transpeptidase, Na+K+ATPase, sucrase and lactase enzymes. But, exposure of membrane vesicles to solubilized ICP's in vitro, lead to significant reduction in the activity of Na+K+ATPase, sucrase and lactase enzymes.

Cloning, expression and toxicity of a mosquitocidal toxin gene of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vitro excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested although the toxicity was not as high as one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot.

Effect of insecticidal crystal proteins of Bacillus thuringiensis on human erythrocytes in vitro.

Effect of intact and alkali solubilized insecticidal crystal protein (ICP) preparations from a mutant strain of B. thuringiensis var. israelensis (VCRC MB24) and the wild type strain (VCRC B17) in vitro on human erythrocytes with respect to lipid peroxidation, osmofragility and membrane bound enzymes was determined. The alkali solubilized ICPs of both B. thuringiensis strains caused increased lipid peroxidation, decreased resistance to hypotonic lysis and reduction in the activity of membrane bound enzymes. On the contrary, the intact ICPs did not produce any such adverse effect on RBCs under the same experimental conditions. It is suggested that the ICPs are safe when they are intact when compared with solubilized ones.

Effect of inorganic salts, soaps and detergents on dissolution and larvicidal activity of alginate formulation of Bacillus sphaericus.

Various inorganic salts and commonly used soaps and detergents were tested in the laboratory for their effect on the dissolution and larvicidal residual activity of a slow-release alginate encapsulated granular formation of Bacillus sphaericus. Fluoride, chloride and sulphate salts and a detergent powder affected the residual activity of this formulation drastically by rupturing it but did not effect its larvicidal activity. Nitrates and phosphates of sodium and potassium also had the same effect but to a moderate level. The safest concentration of these water impurities for effective functioning of the alginate encapsulated B. sphaericus formulation have been determined.

Studies of the antifungal compounds produced by Erwinia herbicola.

The organic phase of a wide spectrum, antimycotic and diffusable toxin from Erwinia herbicola showed a highly significant inhibitory activity against Pyricularia oryzae spores in spore well bioassay. Germ tube lengths were inhibited more in wells containing 5 microliters equivalent of bacterial toxin than 1 microliter. No significant difference between the germ tube in an equal mixture of Dimethyl sulphoxide: ethanol and controls. Thin layer chromatography using the chloroform extraction of the organic phase showed a significant antagonism on Cladosporium cucumerinum. The retardation factor values for inhibitory zones in solvent 1 were 0.07 for lower spot and 0.26 for upper spot.