Phage typing of Bacillus subtilis and B. thuringiensis.

Phage typing schemes for Bacillus subtilis and B. thuringiensis were constructed using 98 phages and 743 bacterial strains. Most phages were host-species-specific. Phages were classified by electron microscopy. The B. subtilis scheme includes 10 phages and 29 phage types. The B. thuringiensis scheme comprises 8 phages and 25 phage types and can be applied to B. cereus. There is no correlation between H antigen serotypes and phagovars in B. thuringiensis. Characteristics of typing phages are described for identity control.

Clostridium bifermentans serovar malaysia: characterization of putative mosquito larvicidal proteins.

The toxicity of Clostridium bifermentans serovar malaysia to mosquito larvae is due to protein toxins, belonging to a novel class of insecticidal toxins. Toxic extracts contains three major proteins of 66, 18 and 16 kDa. The 18-kDa and 16-kDa proteins are probably involved in toxicity. They are synthesised during sporulation, concomitant with activity. They are absent from non-toxic strains of C. bifermentans and are present at very low levels in non-toxic C. bifermentans serovar malaysia cultures produced at 42 degrees C. The 66-kDa protein is present throughout the growth phases of C. bifermentans serovar malaysia, and an immunologically related 66-kDa protein is present in non-toxic C. bifermentans strains.

Vertebrate safety of Clostridium bifermentans serovar malaysia, a new larvicidal agent for vector control.

The safety of bacterial cells of Clostridium bifermentans serovar malaysia, which is highly toxic to mosquito larvae, was tested on mice, guinea pigs, rabbits, and goldfish. Inoculations of at least 1 x 10(8) cells per animal by routes recommended by World Health Organization (subcutaneous, percutaneous, inhalation, force-feeding, intraperitoneal, intravenous) and tests of subacute toxicity, anaphylactic shock, persistence in heart blood, and virulence by successive passages, were performed on mice, guinea pigs, or both. Growth was monitored for 1 mo before necropsy. Ocular irritation and skin scarification were tested with rabbits. C. bifermentans serovar malaysia did not induce any mortality or abnormal reactions in mammals at a dose 1,000 times higher than the level established by W.H.O. for the demonstration of safety. Bacterial cells are not toxic to goldfish at a dose 1,000 times higher than the LD50 for the target-mosquito larvae. We conclude that C. bifermentans serovar malaysia bacterial cells are safe for laboratory mammals and goldfish.

Host range of Clostridium bifermentans serovar. malaysia, a mosquitocidal anaerobic bacterium.

Clostridium bifermentans serovar. malaysia (C.b.m.) is toxic to mosquito larvae. In this study, we quantified its toxicity to the mosquitoes, Aedes aegypti, Ae. albopictus, Ae. caspius, Ae. detritus, Anopheles stephensi, An. gambiae, Culex pipiens and Cx. quinquefasciatus. Anopheles larvae are the most susceptible, followed by Ae. detritus and Ae. caspius, then Culex and other Aedes larvae. According to mosquito species, the LC50 varies from 7 x 10(3) to 1.3 x 10(6) cells/ml. Three concentrations (10(7), 10(6) and 10(5) cells/ml) of C.b.m., Bacillus thuringiensis var. israelensis (B.t.i.) and Bacillus sphaericus were tested on Ae. aegypti, An. stephensi and Cx. pipiens larvae in order to determine the time necessary for each concentration to kill 50 and 90% of the population. Ninety percent of the 3 mosquito populations are killed within 4-15 h by the C.b.m. concentrations. Whatever the concentrations, C.b.m. kills at least 10 times less rapidly than B.t.i. but always quicker than B. sphaericus. Bioassays of C.b.m. bacterial cells or final whole culture were not toxic to Musca domestica and Drosophila melanogaster (Diptera) as well as to Phaedon cochleariae (Coleoptera) and Spodoptera littoralis (Lepidoptera).

A new serotype of Bacillus thuringiensis from Colombia toxic to mosquito larvae.

During a survey conducted in Colombia a new isolate of Bacillus thuringiensis that showed toxicity toward Culex quinquefasciatus, Cx. pipiens, Aedes aegypti, and Anopheles stephensi larvae was isolated. Parasporal crystals were spherical in shape and showed a great degree of similarity with those produced by the reference strain of Bacillus thuringiensis subsp. israelensis. Supernatant fraction of the whole culture was not toxic, and heat-stable exotoxin production was negative. Catalase, urease, arginine dihydrolase, amylase, lecithinase, acetyl-methyl-carbinol, and gelatinase production were positive. Hemolysis on sheep blood agar was alpha-type. The isolate 163-131 showed natural resistance to azolocillin and was sensible to cephoperazone, cephalotin, nalidixic acid, and trimetoprin sulfametoxazole. Flagellar agglutination showed a specific H 30 antigen which allows individualization of this strain as a new serotype and the subspecies name of medellin is proposed.

Two novel strains of Bacillus thuringiensis toxic to coleopterans.

Two novel strains of Bacillus thuringiensis were isolated from native habitats by the use of genes coding for proteins toxic to coleopterans (cryIII genes) as hybridization probes. Strain EG2838 (isolated by the use of the cryIIIA probe) contained a cryIIIA-hybridizing plasmid of approximately 100 MDa and synthesized crystal proteins of approximately 200 (doublet), 74, 70, 32, and 28 kDa. Strain EG4961 (isolated by the use of a cryIIIA-related probe) contained a cryIIIA-hybridizing plasmid of approximately 95 MDa and synthesized crystal proteins of 74, 70, and 30 kDa. Structural relationships among the crystal proteins of strains EG2838 and EG4961 were detected; antibodies to the CryIIIA protein toxic to coleopterans reacted with the 74- and 70-kDa proteins of EG2838 and EG4961, antibodies to the 32-kDa plus 28-kDa proteins of EG2838 reacted with the 30-kDa protein of EG4961, and antibodies to the 200-kDa proteins of EG2838 reacted with the 28-kDa protein of EG2838. Experiments with B. thuringiensis flagella antibody reagents demonstrated that EG2838 belongs to H serotype 9 (reference strain B. thuringiensis subsp. tolworthi) and that EG4961 belongs to H serotype 18 (reference strain B. thuringiensis subsp. kumamotoensis). A mixture of spores plus crystal proteins of either EG2838 or EG4961 was toxic to the larvae of Colorado potato beetle (Leptinotarsa decemlineata), and significantly, the EG4961 mixture was also toxic to the larvae of southern corn rootworm (Diabrotica undecimpunctata howardi). DNA restriction blot analysis suggested that strains EG2838 and EG4961 each contained a unique gene coding for a protein toxic to coleopterans.

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.

Clostridium bifermentans serovar malaysia: sporulation, biogenesis of inclusion bodies and larvicidal effect on mosquito.

Sporulation of Clostridium bifermentans serovar malaysia, which has a larvicidal activity towards mosquitoes, was examined by electron microscopy. Parasporal inclusion bodies lacking a crystalline structure were first detected at t5 (5 h after the end of exponentional growth). Also, the presence of "brush-bottle"-like appendages appearing first at t5 was noted; these remained attached to the spores when released after sporangium lysis. Larvicidal activity assayed on Anopheles stephensi larvae appeared at t0 and increased rapidly to a maximum between t5 and t8. However, a decrease in bacterial toxicity occurred with sporangium lysis.

[Clostridium bifermentans serovar malaysia, a new anaerobic bacterium pathogen to mosquito and blackfly larvae]. / Clostridium bifermentans serovar malaysia, une nouvelle bactérie anaérobie pathogène des larves de moustiques et de simulies.

A strain of Clostridium bifermentans individualized as serovar malaysia (C.b.m.) according to its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity occurs in sporulated cells which contain, in addition to spores, proteinic parasporal inclusion bodies and feather-like appendages; the amino acid content of the inclusion bodies is similar to that of Bacillus thuringiensis serovar israelensis (B.t.i.) and B. sphaericus crystals. The toxicity to Anopheles stephensi is as high as that of B.t.i. and the best strains of B. sphaericus. Culex pipiens is somewhat less susceptible, and Aedes aegypti much less. Pure parasporal inclusion bodies, isolated by ultracentrifugation on sucrose gradients, are highly toxic to mosquito larvae. The larvicidal power is destroyed by heating at 80 degrees C or by treatment with 50 mM NaOH. It is preserved by freeze-drying. The innocuity to mice of the sporulated cells is shown by different routes of administration: force-feeding, percutaneous, subcutaneous, intraperitoneal or intravenous injections. The potential for the biological control of mosquito and blackfly larvae is suggested.

Another Bacillus sphaericus serotype harbouring strains very toxic to mosquito larvae: serotype H6.

Ten isolates of Bacillus sphaericus from Ghana, very toxic to mosquito larvae, have been identified as belonging to serotype H6. These isolates can be represented by the head-group strain IAB59. They form crystals at the sporulation stage. Their larvicidal effect on Culex pipiens and Anopheles stephensi larvae is as high as that of the most toxic strains already known, e.g. 1593 and 2362 (serotype H5a,5b) and 2297 (serotype H25). Spore-crystal extracts of all these strains contain a 43-Kd polypeptide immunologically related to the 43-Kd polypeptide from strain 2362 described by other authors.

Bacillus sphaericus asporogenous mutants: morphology, protein pattern and larvicidal activity.

Asporogenous mutants from Bacillus sphaericus strains 2297 and 1593-4, blocked at different stages of the sporulation process, were isolated. Two mutants (2297 Aspo30A and 2297 Aspo34) which were blocked early in sporulation did not possess any crystalline inclusions and were poorly toxic to Culex pipiens mosquito larvae. Other mutants (2297 Aspo115, 2297 Aspo24 and 1593-4 Aspo12) which were blocked at later stages synthesized crystal-like inclusions next to the forespores, and were highly toxic to mosquito larvae. Electrophoretic protein analysis of alkali extracts from mutants and wild type strains confirmed the absence of toxic crystal-related proteins in early-blocked mutants and their presence in later ones. Western blots with antisera directed against the crystal proteins confirmed those observations.

Sporulation of Bacillus sphaericus 2297: an electron microscope study of crystal-like inclusion biogenesis and toxicity to mosquito larvae.

Sporulation of Bacillus sphaericus strain 2297 in a synchronous liquid culture was studied by electron microscopy. The t0 of sporulation occurred 7 h after the beginning of the lag phase. Crystal-like inclusions first appeared at t2 and reached their final size between t5 and t6. The release of the spore/inclusion complex occurred at about t15 (22 h after inoculation). Toxicity against Culex pipiens larvae was related to sporulation and appeared during the early stages of sporulation. The LC50 (24 h) decreased about 10(5)-fold between t0-2 and t7, in correlation with the formation of crystalline inclusions. Heat resistance of spores appeared later than toxicity.

[Action of crystals of Bacillus thuringiensis var. israelensis on the midgut of Aedes aegypti L. larvae, studied by electron microscopy]. / Action des cristaux de Bacillus thuringiensis var. israelensis sur l'intestin moyen des larves de Aedes aegypti L., en microscopie électronique.

Ingestion of Bacillus thuringiensis var. israelensis crystals by Aedes aegypti larvae is followed by midgut epithelium disruption. Earliest ultrastructural changes consist of an enlargement of intra- and intercellular spaces in the basal region of the cell. Endoplasmic reticula disintegrate by forming spherical structures which increase in size during intoxication. Mitochondria are transformed at first into a condensed form, then become swollen with the disappearance of internal cristae. In the cardia cells, which secrete the peritrophic membrane, the Golgi apparatus may produce electron-dense secretion vesicles; in this event, the peritrophic membrane assumes an abnormal configuration. Before complete breakdown, a cellular hypertrophy is observed: few cells become balloon-like. At the same time, the microvilli decrease in size, widen and then disappear after a few hours exposure to crystal delta-endotoxin.

[Electron microscope study of "Bacillus thuringiensis" var. "Israelensis" sporulation and crystal biogenesis (author's transl)]. / Sporulation et cristallogenèse de Bacillus thuringiensis var. Israelensis en microscopie électronique.

The sporulation and crystal development of Bacillus thuringiensis serotype H-14 was described for a wild spore- and crystal-forming strain and for a mutant crystal but non spore-forming strain. The special nature of the israelensis var. consisted in the composite structure of the crystal, made of a number of components differing in size, shape and electron density. The components were formed in a single inclusion or sometimes separately inside the same bacterial cell.