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Cats represent a potential source of Coxiella burnetii, the aetiological agent of Q fever in humans. The prevalence and risk factors of C. burnetii infection in farm, pet and feral cats were studied in Quebec, Canada, using a cross-sectional study. Serum samples were tested using a specific enzyme-linked immunosorbent assay (ELISA) for the presence of antibodies against C. burnetii, whereas rectal swabs were assayed using real-time quantitative polymerase chain reaction (qPCR) for the molecular detection of the bacteria. Potential risk factors for farm cats were investigated using clinical examinations, questionnaires and results from a concurrent study on C. burnetii farm status. A total of 184 cats were tested: 59 from ruminant farms, 73 pets and 52 feral cats. Among farm cats, 2/59 (3.4%) were ELISA-positive, 3/59 (5.1%) were ELISA-doubtful and 1/59 (1.7%) was qPCR-positive. All pets and feral cats were negative to C. burnetii ELISA and qPCR. Farm cat positivity was associated with a positive C. burnetii status on the ruminant farm (prevalence ratio = 7.6, P = 0.03). Our results suggest that although pet and feral cats do not seem to pose a great C. burnetii risk to public health, more active care should be taken when in contact with cats from ruminant farms.

Background: Tail-biting (TB) is a serious behavioral disorder in pigs. It is defined as a pig chewing the tail of another pig. It is an important challenge in swine production as it impacts the animal welfare, its health and the economics and safety of the pork meat supply chain. Some treatments have been proposed but have not proven optimal. Nutrition, feed type and composition, appears to be an important factor in TB behavior, perhaps by modulating the intestinal microbiota (IM). In humans, IM is increasingly recognized as a modulator of behavior. Our aim was to assess an association between TB behavior and IM in pigs through comparisons of IM in groups of biter or bitten pigs to the IM in a non-biter and non-bitten negative control group, and through comparisons of IM between the biter and bitten groups. Each group, composed of 12 pigs, was formed at the beginning of the growing/finishing phase based on a target behavior analysis centered on TB behavior for the biter group and a score of damages caused to the tail for the bitten group. Fecal and blood samples were collected from each pig during a TB episode, at time 0, t0, and after a TB episode, four weeks later, at time 1, t1. Serum cortisol level was determined by ELISA and used as an indicator of stress. The pig’s fecal microbiota was analyzed from DNA extracted from freshly collected fecal matter using amplicon sequencing of the V4 hypervariable region of the 16S rRNA gene. Results: Serum cortisol levels were significantly higher in either the biter or bitten pig groups compared to the negative control group ( p =0.02 and p =0.01, respectively). Interestingly, no significant difference was revealed between the biter and bitten groups. The microbiota alpha-diversity was not significantly different between all groups, biter, bitten and negative control. Analyses of beta-diversity, however, revealed a significant difference between either the biter or the bitten group in comparison to the negative control group in terms of structure and composition of the microbiota. Lactobacillus were significantly more abundant in the negative control group compared to the two other groups ( p =0.001). No significant difference was revealed between the biter and bitten groups. Quantitative real-time PCR (qPCR) confirmed that lactobacilli were more abundant in the negative control group. Conclusions: This study demonstrates that TB behavior is associated to the IM composition in pigs.

Purpose. Respiration-induced motion introduces significant positioning uncertainties in radiotherapy treatments for thoracic sites. Accounting for this motion is a non-trivial task commonly addressed with surrogate-based strategies and latency compensating techniques. This study investigates the potential of a new unified probabilistic framework to predict both future target motion in real-time from a surrogate signal and associated uncertainty. Method. A Bayesian approach is developed, based on a Kalman filter theory adapted specifically for surrogate measurements. Breathing motions are collected simultaneously from a lung target, two external surrogates (abdominal and thoracic markers) and an internal surrogate (liver structure) for 9 volunteers during 4 min, in which severe breathing changes occur to assess the robustness of the method. A comparison with an artificial non-linear neural network (NN) is performed, although no confidence interval prediction is provided. A static worst-case scenario and a simple static design are investigated. Results. Although the NN can reduce the prediction errors from thoracic surrogate in some cases, the Bayesian framework outperforms in most cases the NN when using the other surrogates: bias on predictions is reduced by 38% and 16% on average when using respectively the liver and the abdomen for the simple scenario, and by respectively 40% and 31% for the worst-case scenario. The standard deviation of residuals is reduced on average by up to 42%. The Bayesian method is also found to be more robust to increasing latencies. The thoracic marker appears to be less reliable to predict the target position, while the liver shows to be a better surrogate. A statistical test confirms the significance of both observations. Conclusion. The proposed framework predicts both the future target position and the associated uncertainty, which can be valuably used to further assist motion management decisions. Further investigation is required to improve the predictions by using an adaptive version of the proposed framework.

Objective To evaluate whether pig farms interconnected within the same cooperative share similar Salmonella contamination patterns. Setting Ten finishing pig farms within a 100 km radius of a common slaughterhouse were selected. Their inclusion was based on their association to the same cooperative and the sharing of common resources: piglets, feed, swine transporters, slaughterhouse, technicians and veterinarians. Procedure Each farm was visited three times over a 10-month period. Pig faeces, the barn front door handle, the feed pipeline, mobile objects (shovel, balance and pig board), the landing stage, the concrete slab of the feed bins, the tire tracks left on the pathways by the animal feed truck, the pig delivery truck and the carcase knacker truck and the mudguards and cabin carpets of the veterinarian and technician vehicles on their arrival at the farm were all analysed for the presence of Salmonella. Results All farms were not equally contaminated with Salmonella. Whereas some farms yielded up to 12 Salmonella isolates, other farms were Salmonella free. Some locations, most notably the landing stage, were more contaminated than others. Salmonella contamination was dynamic in time. Some contaminations seen on farms, on specific locations on the first visit, had disappeared on the second and third visits, but new contaminations were detected on different locations. Conclusions Contamination with Salmonella was not disseminated through the network of the 10 pig farms interconnected within the same cooperative but was rather most often restricted in time to specific locations on specific farms.

This report describes various Salmonella serovars which were found on often overlooked locations in a pig farm/slaughterhouse interface. These include slaughterhouse yard pathways and mudguards and carpets of transport trucks arriving at and departing from production sites.

Listeria monocytogenes is the etiological agent of listeriosis, a major foodborne disease and an important public health concern. Contamination of meat with L. monocytogenes occurs frequently at the slaughterhouse. Our aims were; 1) to investigate the distribution of L. monocytogenes in the processing areas of four swine slaughterhouses; 2) to describe the diversity of L. monocytogenes strains by pulsed-field gel electrophoresis; 3) to identify persistent L. monocytogenes strains and describe their distribution; 4) to investigate the associations between persistence of strains and their following characteristics: detection in food isolates, detection in human clinical isolates, and the presence of benzalkonium chloride (BAC) resistance genes. Various operation areas within the four swine slaughterhouses were sampled on four occasions. A total of 2496 samples were analyzed, and L. monocytogenes was successfully isolated from 243 samples. The proportion of positive samples ranged from 32 to 58% in each slaughterhouse and from 24 to 68% in each operation area. Fifty-eight different pulsotypes were identified and eight pulsotypes, present in samples collected during 4 visits, were considered persistent. The persistent pulsotypes were significantly more likely to be detected in food (P < 0.01, exact χ²) and human clinical cases (P < 0.01, exact χ²), respectively. Among pulsotypes harboring the BAC bcrABC resistance cassette or the emrE multidrug transporter gene, 42.8% were persistent compared to 4.5% for pulsotypes without these resistance genes (P < 0.01, exact χ²). Our study highlights the importance of persistent L. monocytogenes strains in the environmental contamination of slaughterhouses, which may lead to repeated contamination of meat products. It also shows that the presence of disinfectants resistance genes is an important contributing factor.

Biofouling, the accumulation of microorganisms, is a major problem in paper mills processing paper and cardboard. This leads to the production of lower quality recycled products. Several studies have focused on the microbial content in the paper mill and the final products. Our aim was to determine the microbial biota in a bale of collected cardboard prior to entering the paper mill. Total genomic DNA was isolated and analyzed using two different methods for comparison purposes: 454 pyrosequencing and clone library. A total of 3268 V6-V8 454 pyrosequencing reads and 322 cloned V6-V8 16S rRNA nucleotide sequences were obtained. Both methods showed the presence of three major bacterial genera: Bacillus, Solibacillus and Paenibacillus, all members of the spore-forming phylum Firmicutes. Pyrosequencing, however, revealed a richer and more diverse bacterial community than clone library. It showed the presence of additional minor Firmicute genera and of a small number of Proteobacteria. The sorting at the recycling plant, the storing, and the processing at the paper mill, the end uses, will all contribute to the bacterial microbiota present in a bale of collected cardboard as revealed here.

The whole genome sequencing of extensively drug-resistant Shewanella xiamenensis T17 isolated from hospital effluents in Algeria revealed the presence of a novel 268.4 kb plasmid designated pSx1, which carries several antibiotic-resistance genes in the novel Tn1696 derivative (Tn6297), in addition to the chromosomal blaOXA-48-like gene (blaOXA-416). The presence of the plasmid was confirmed by nuclease S1-PFGE analysis and transformation by electroporation into Escherichia coli DH10B. Tn6297 contains an In27 class 1 integron harboring the dfrA12-orfF-aadA2 array, msr(E) and mph(E) associated with IS26; a new efflux pump multidrug resistance composite transposon delimited by two ISEc29s; Tn-tet harboring tetR and tetA(C); a class 1 integron with the qacG gene cassette; qnrVC6 and dfrA23 associated with ISCR1; and a complex class 1 integron In4-like containing aacC1, aadA1, blaVEB-16, catA2, sul1Δ, cmlA9, tetR, tetA(G), aac(6′)-II, and blaPSE-1. Its mer operon carries merB, but lacks merC, in contrast to Tn...

Necrotizing fasciitis is a serious disease characterized by the necrosis of the subcutaneous tissues and fascia.E. colias the etiologic agent of necrotizing fasciitis is a rare occurrence. A 66-year-old woman underwent total abdominal hysterectomy with bilateral salpingo-oophorectomy. She rapidly developed necrotizing fasciitis which led to her death 68 hours following surgery. AnE. colistrain was isolated from blood and fascia cultures. DNA microarray revealed the presence of 20 virulence genes.

A short phylogenetic marker previously used in the reconstruction of the Class γ-proteobacteria was assessed here at a lower taxa level, species in the genus Xanthomonas. This maker is 224 nucleotides in length. It is a combination of a 157 nucleotide sequence at the 3' end of the 16S rRNA gene and a 67 nucleotide sequence at the 5' end of the 16S-23S ITS sequence. A total of 23 Xanthomonas species were analyzed. Species from the phylogenetically related genera Xylella and Stenotrophomonas were included for com- parison purposes. A bootstrapped neighbor- joining phylogenetic tree was inferred from comparative analyses of the 224 bp nucleotide sequence of all 30 bacterial strains under study. Four major Groups were revealed based on the topology of the neighbor-joining tree, Group I to IV. Group I and II contained the genera Steno-trophomonas and Xylella, respectively. Group III included five Xanthomonas species: X. theicola, X. sacchari, X. albineans, X. transluscens and X. hyacinthi. This group of Xanthomonas species is often referred to as the hyacinthi group. Group IV contained the other 18 Xanthomonas species. The overall topology of the neighbor-joining tree was in agreement with currently accepted phylogenetic. The short phylogenetic marker used here could resolve species from three dif-ferent Xanthomonadacea genera: Stenotro-phomonas, Xylella and Xanthomonas. At the level of the Xanthomonas genus, distant spe-cies could be distinguished, and whereas some closely-related species could be distinguished, others were undistinguishable. Pathovars could not be distinguished. We have met the resolving limit of this marker: pathovars and very closely related species from same genus.

A short 220 bp sequence was used to study the taxonomic organization of the bacterial Order Bacillales. The nucleotide sequences of the 3’ end of the 16S rDNA and the 16S-23S Internal transcribed spacer (ITS) were determined for 32 Bacillales species and strains. The data for 40 additional Bacillales species and strains were retrieved directly from Genbank. Together, these 72 Bacillales species and strains encompassed eight families and 21 genera. The 220 bp se- quence used here covers a conserved 150 bp sequence located at the 3’ end of the 16S rDNA and a conserved 70 bp sequence located at the 5’ end of the 16S-23S ITS. A neighbor-joining phylogenetic tree was inferred from comparative analyses of all 72 nucleotide sequences. Eight major Groups were revealed. Each Group was sub-divided into sub-groups and branches. In general, the neighbor-joining tree presented here is in agreement with the currently accepted phylogeny of the Order Bacillales based on phenotypic and genotypic data. The use of this 220 bp sequence for phylogenetic analyses presents several advantages over the use of the entire 16S rRNA genes or the generation of extensive phenotypic and genotypic data. This 220 bp sequence contains 150 bp at the 3’ end of the 16S rDNA which allows discrimination among distantly related species and 70 bp at the 5’ end of the 16S-23S ITS which, owing to its higher percentage of nucleotide sequence divergence, adds discriminating power among closely related species from same genus and closely related genera from same family. The method is simple, rapid, suited to large screening programs and easily accessible to most laboratories.

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3’ end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5’ end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3’ end of the 16S rRNA gene and 75 bp from the 5’ end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

A short phylogenetic marker previously used in the reconstruction of the Order Bacillales and the genus Bacillus was assessed here at a lower taxa level: species in the Bacillus cereus group: B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis. This maker is 220 bp in length. It is a combination of 150 bp at the 3’ end of the 16S rDNA and 70 bp at the 5’ end of the 16S-23S ITS sequence. Three additional Bacillus species, B. halodurans, B. licheniformis and B. subtilis, and Clostridium tetani were included for comparison purposes. A total of eight bacterial species and 12 strains were analyzed. A boot- strapped neighbor-joining tree was inferred from comparative analyses of all allelic sequences of the bacterial species and strains under study. Based on its topology, four major Groups were revealed at the 90% nucleotide sequence identities, Group I to IV. Group I contains all al-leles of the Bacillus cereus group. Group II con-tains all alleles of B. halodurans. Group III con-tains all alleles of B. licheniformis and B. subtilis. Group IV contains all alleles of Clostridium tetani. The 220 bp phylogenetic marker used here could resolve different species from different genera. At the genus level, distant species could be dis-tinguished. Very closely-related species, however, were undistinguishable. Species in the B. cereus group, most notably B. cereus, B. anth- racis and B. thuringiensis, could not be distin- guished. After successfully inferring the phylo- genies of the Order Bacillales and the genus Bacillus, we have met the resolving limit of this short phy-logenetic marker: B. cereus, B. anthracis and B. thuringiensis.

Aims: To characterize a novel, unusual, Bacillus thuringiensis strain, to clone its cry gene and determine the spectrum of action of the encoded Cry protein. Methods and Results: The B. thuringiensis strain, referred to as M15, was isolated from dead two-spotted spider mites (Tetranychus urticae Koch; Arthropoda: Arachnida: Tetranychidae). It is an autoagglutination-positive strain and is therefore non-serotypeable. A sporulated culture produces a roughly spherical parasporal inclusion body, the crystal, tightly coupled to the spore. Although the crystal appears to be composed of at least two major polypeptides of 86 and 79 kDa as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Southern hybridization indicates that the corresponding crystal protein gene is likely present in only one copy. The crystal protein gene was cloned and, based on nucleotide sequence homology with an orthologous cry31Aa1 gene, assigned the name cry31Aa2. Although initially isolated from spider mites, B. thuringiensis M15 is non-toxic to spider mites and it does not produce the wide spectrum β-exotoxin. Assays on mammalian cells, however, reveal that Cry31Aa2, when cleaved with trypsin, is cytocidal to some human cancer cells but not to normal human cells. No cytocidal activity was induced after protease treatment of Cry31Aa2 with either chymotrypsin or proteinase K. Trypsin, chymotrypsin and proteinase K cleavage sites were determined. Conclusions: The B. thuringiensis strain M15 exhibits specific cytocidal activities against some human cancer cells. Significance and Impact of the Study: This study raises questions as to the actual role of this bacterial strain and its crystal protein in the environment. It may be possible to further develop the Cry31Aa2 protein to target specific human cancer cells.

A 5.7-kb EcoRI fragment was cloned from plasmid DNA of Bacillus thuringiensis strain M15. It contains two insertion sequences (IS), IS231M2 and -M1 in the 5'-3' order, arranged in tandem, in same orientation, separated by a 540-bp region. The primary structure is typical of a composite transposon, here of 3847 bp in length, for which the name Tn231M is proposed. Each IS is delimited by 18-bp inverted repeats (IR), and flanked by 11-bp direct repeats (DR). Both IS share 99.3% nucleotide identities. IS231M1 has a single open reading frame (ORF) which encodes a putative 477-amino-acid transposase. IS231M2 has two smaller ORFs: ORF1 and ORF2, which could code for polypeptides of 329 and 118 amino acids in length, respectively. Further analysis reveals that the regions upstream of IS231M2, and downstream of -M1, and the 540-bp region, contain additional pairs of IR and DR. Interestingly, potential annealing between all pairs of IR and DR could generate two unusual cloverleaf secondary structures.

Here, we present the draft genome sequence of a necrotoxigenic Escherichia coli strain isolated from a patient following a very rapidly evolving, lethal necrotizing fasciitis.

Here, we present the draft genome sequences of two toxigenic Corynebacterium ulcerans strains isolated from two different patients: one from a blood sample and the other from a scar exudate following surgery. Although these two strains harbor the diphtheria toxin gene tox , no full prophage sequences were found in the flanking regions.

We set out to analyze the sequence diversity of the Bacillus thuringiensis flagellin (H antigen [Hag]) protein and compare it with H serotype diversity. Some other Bacillus cereus sensu lato species and strains were added for comparison. The internal sequences of the flagellin ( hag ) alleles from 80 Bacillus thuringiensis strains and 16 strains from the B. cereus sensu lato group were amplified and cloned, and their nucleotide sequences were determined and translated into amino acids. The flagellin allele nucleotide sequences for 10 additional strains were retrieved from GenBank for a total of 106 Bacillus species and strains used in this study. These included 82 B. thuringiensis strains from 67 H serotypes, 5 B. cereus strains, 3 Bacillus anthracis strains, 3 Bacillus mycoides strains, 11 Bacillus weihenstephanensis strains, 1 Bacillus halodurans strain, and 1 Bacillus subtilis strain. The first 111 and the last 66 amino acids were conserved. They were referred to as the C1 and C2 regions, respectively. The central region, however, was highly variable and is referred to as the V region. Two bootstrapped neighbor-joining trees were generated: a first one from the alignment of the translated amino acid sequences of the amplified internal sequences of the hag alleles and a second one from the alignment of the V region amino acid sequences, respectively. Of the eight clusters revealed in the tree inferred from the entire C1-V-C2 region amino acid sequences, seven were present in corresponding clusters in the tree inferred from the V region amino acid sequences. With regard to B. thuringiensis , in most cases, different serovars had different flagellin amino acid sequences, as might have been expected. Surprisingly, however, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. Likewise, serovars from the same H serotypes were most often found clustered together, with exceptions. Indeed, some serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. Species-wise, B. halodurans , B. subtilis , and B. anthracis formed specific branches, whereas the other four species, all in the B. cereus sensu lato group, B. mycoides , B. weihenstephanensis , B. cereus , and B. thuringiensis , did not form four specific clusters as might have been expected. Rather, strains from any of these four species were placed side by side with strains from the other species. In the B. cereus sensu lato group, B. anthracis excepted, the distribution of strains was not species specific.

Nucleotide sequence comparisons of three house-keeping genes, adenylate kinase (adk), shikimate dehydrogenase (aroE), and glucose-6-phosphate dehydrogenase (gdh), were used to infer the phylogeny of 33 gamma-proteobacteria. Phylogenetic trees inferred from each gene, and from the concatenated sequences of all three genes, are, in general, similar to a 16S rRNA gene-inferred tree. Similar grouping of bacteria are revealed at the family, genus, species and strain levels in all five trees. The house-keeping genes, however, show a higher rate of nucleotide sequence substitutions. Consequently, they can possibly probe deeper branches of a phylogenetic tree than the 16S rRNA gene. However, because their nucleotide sequences are not as highly conserved among gamma-proteobacteria, family- or genus-specific primers would need to be designed for the amplification of any of these three house-keeping genes. Since these genes are used in multilocus sequence typing, it is expected that the number of sequences publicly available for many taxa will increase over time proving them very useful either at complementing 16S rRNA-inferred phylogenies or for specific, targeted, phylogenetic analysis.

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium that has been developed for the control of some insect pests in the orders Lepidoptera, Coleoptera, and Diptera. In this study, whole culture extracts of 94 B. thuringiensis strains from 83 serovars were added to an artificial diet and assayed against L. hesperus first and second instars. A total of five B. thuringiensis strains, B. thuringiensis variety thuringiensis, thuringiensis exotoxin +, morrisoni, tolworthi, and darmstadiensis generated98% mortality after 7 d of incubation. The screening was repeated with 117 alkali-solubilized trypsin-digested B. thuringiensis cultures and the same five B. thuringiensis strains showed nearly identical results. All five strains produce beta-exotoxin, which exhibits a wide host spectrum activity. No beta-exotoxin-minus B. thuringiensis strains showed significant toxicity against L. hesperus nymphs. The present work is one of the first thorough screenings of the wide diversity of the B. thuringiensis varieties for the control of L. hesperus nymphal populations.

The nucleotide sequences of the 3' end of the 16S rDNA and the 16S-23S internal transcribed spacer (ITS) of 40 Bacillaceae species were determined. These included 21 Bacillus, 9 Paenibacillus, 6 Brevibacillus, 2 Geobacillus, 1 Marinibacillus and 1 Virgibacillus species. Comparative sequence analysis of a 220 bp region covering a highly conserved 150 bp sequence located at the 3' end of the 16S rRNA coding region and a conserved 70 bp sequence located at the 5' end of the 16S-23S ITS of the 40 species and six sequences available in GenBank were used to infer the phylogenetic relationships between all 46 taxa. When a maximal distance (D(max), where D refers to the number of nucleotide substitutions per site) of 0.31 was introduced as a threshold to determine groupings, 10 phylogenetically distinct clusters were revealed. Twenty-six Bacillus species were separated in seven groups (I, II, III, IV, V, VI and X), but Bacillus circulans remained ungrouped. All six Brevibacillus species under study were in Group VII. The nine Paenibacillus species fell into two distinct groups (VIII and IX). Species with D(max) values within 0.05 were considered to be very closely related. These were Bacillus psychrophilus and Bacillus psychrosaccharolyticus in Group II; 'Bacillus maroccanus' and Bacillus simplex in Group II; Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus mojavensis and Bacillus subtilis in Group VI; Bacillus fusiformis and Bacillus sphaericus in Group VI; Brevibacillus brevis and Brevibacillus formosus in Group VII; Paenibacillus gordonae and Paenibacillus validus in Group VIII; and Bacillus anthracis, Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis in Group X. The phylogenetic classification presented here is, in general, in agreement with current classifications based on phenotypic and molecular data. Our findings suggest, however, that in some cases, further divisions or, conversely, further groupings might be warranted. Should current classifications be re-examined in the light of our results, D(max) values of 0.31 and 0.05, as exemplified here, may prove useful threshold values for the grouping of Bacillaceae into taxa akin to genera and species, respectively. These D(max) thresholds may also reveal, in a different way, bacterial species for which further characterization might be warranted for proper classification and/or reassignment.

Twenty-three isolates of Beauveria bassiana (Balsamo) Vuillemin from different hosts and geographical origins were evaluated under laboratory conditions for various aspects of virulence against 2- to 3-d-old third instars of the obliquebanded leafroller, Choristoneura rosaceana (Harris), a pest of apple, Malus domestica Borkhausen (Rosaceae), and several other orchard crops in North America. When submerged in a solution containing 107 conidia/mL, all B. bassiana isolates caused more than 66% larval and pupal mortality 60 d following treatment. Isolates 14, 16, 1525, 2727, and 2990 caused the highest (80–89%) larval and pupal mortality against C. rosaceana. Relative to the control, isolate 63 significantly increased and isolate 44860 significantly decreased larval development time. Isolates 37, 2727, and 2990 significantly decreased C. rosaceana pupal weight. Isolates 139, 2727, and 2990 significantly decreased the number of emerging females. In view of their larvicidal effects, as well as their carry-over effects on pupae and adult females, isolates 2727 and 2990 showed the best mycoinsecticidal potential against C. rosaceana.

Aims: To identify Bacillus species and related genera by fingerprinting based on ribosomal RNA gene restriction patterns; to compare ribosomal RNA gene restriction patterns-based phylogenetic trees with trees based on 16S rRNA gene sequences; to evaluate the usefulness of ribosomal RNA gene restriction patterns as a taxonomic tool for the classification of Bacillus species and related genera. Methods and Results: Seventy-eight bacterial species which include 42 Bacillus species, 31 species from five newly created Bacillus-related genera, and five species from five phenotypically related genera were tested. A total of 77 distinct 16S rRNA gene hybridization banding patterns were obtained. The dendrogram resulting from UPGMA analysis showed three distinct main genetic clusters at the 75% banding pattern similarity. A total of 77 distinct 23S and 5S rRNA genes hybridization banding patterns were obtained, and the dendrogram showed four distinct genetic clusters at the 75% banding pattern similarity. A third dendrogram was constructed using a combination of the data from the 16S rRNA gene fingerprinting and the 23S and 5S rRNA genes fingerprinting. It revealed three distinct main phylogenetic clusters at the 75% banding pattern similarity. Conclusions: The Bacillus species along with the species from related genera were identified successfully and differentiated by ribosomal RNA gene restriction patterns, and most were distributed with no apparent order in various clusters on each of the three dendrograms. Significance and Impact of the Study: Our data indicate that ribosomal RNA gene restriction patterns can be used to reconstruct the phylogeny of the Bacillus species and derived-genera that approximates, but does not duplicate, phylogenies based on 16S rRNA gene sequences.

A new IS231 variant, IS231N, has been isolated from an autoagglutinable, non-serotypable strain of B. thuringiensis. IS231N is 1654 bp in length and is delimited by two incomplete 20-bp inverted repeats (IRL and IRR) with two mismatches. No direct repeats (DRs) were found at the right and left borders of IS231N. Surprisingly, IS231N contains three open reading frames (ORFs) that could code for polypeptides of 329 (ORF1), 118 (ORF2), and 17 (ORF3) amino acids, respectively. IS231N lacks the 5th conserved amino acid domain, called C2, owing to the addition of an adenine residue at nucleotide 1319. IS231N shows the highest nucleotide identity (99%) with IS231M, another insertion sequence previously isolated from the same bacterial strain. IS231N, however, shares only 83% amino acid identity with IS231M because of nucleotide substitutions and additions. The ORF1 of IS231N has five fewer amino acids than ORF1 of IS231M. Furthermore, the ORF2-3 putative fusion product in IS231N contains eight fewer amino acids than ORF2 in IS231M. The dendrogram showing the evolutionary relationship between members of the IS231 family and IS231N indicates that IS231N is phylogenetically more closely related to IS231M (83%), followed by IS231F(74%), and is more distant from IS231V and W(46%).

EcoRI and HindIII restriction fragment length polymorphism (RFLP) profiles using 2 random DNA probes, named 104 and 106, were generated for 85 B. thuringiensis strains. These include 80 serovars, 4 intra-serovar strains: kurstaki HD-1, dendrolimus, tenebrionis and sandiego, and a non-serotypeable strain B. thuringiensis var. wuhanensis. A total of 47 EcoRI and 65 HindIII restriction patterns were generated when hybridization results from both probes were combined. Seventy-seven B. thuringiensis strains showed distinctive hybridization profiles. The dendrogram resulting from the numerical analysis of the distance matrix revealed fourteen distinct phylogenetic groups at the 96% banding patterns similarity. The intra-serovar strains showed higher similarity with their respective type serovars. However, different serovars from a common H-serotype did not always cluster in the same phylogenetic group. Alternatively, several mosquitocidal serovars clustered in a single phylogenetic group. The correlation between serotyping and banding pattern similarity is discussed.

One hundred and twoBacillus thuringiensisBerliner strains isolated from six different types of Canadian soil and dust from different grain storage bins were cultured in shake flasks containing Great Northern White Bean (GNWB) protein concentrate (48.6% protein) as the main nitrogen source and dextrose as the main carbohydrate source. The resulting endotoxins were bioassayed against the bertha armyworm,Mamestra configurataWlk. Thirty-three percent of soil and 66% of grain dust samples were positive forB. thuringiensis. The bacterium was found most frequently in organic-rich soil. The four most toxic soil isolates (which were seven to 15 times more toxic than the international standard, HD1-S-1980), and two nontoxic grain dust isolates were characterized by serological typing, biochemical analysis, carbohydrate utilization, plasmid profile analysis, protein profile analysis using sodium dodecyl sulfate – polyacrylamide gels, and polymerase chain reaction. Four isolates were determined to be subsp.kurstakicontaining 130–140 and 63–65 kDa proteins, and two isolates (tested for comparison) were subsp.canadensiscontaining 31 and 38 kDa proteins. Nonpyramidal-crystal-producing strains did not grow well in culture media containing GNWB, degossypellized cotton seed meal (61% protein), defatted soy flour (55% protein), or peptone as nitrogen sources. Excess of GNWB protein concentrate in shake flask culture media (30 g/L) inhibited bacterial growth and reduced the toxicity of isolate A1.2/72 subsp.kurstaki, which was the most toxic soil isolate. Isolate A1.2/72, which was 15 times more toxic for bertha armyworm larvae than the international standard (HD1-S-1980), contained threecry1Agenes (cry1Aa,cry1Ab, and cry1Ac), whereas HD-1 lacked thecry1Abgene. This strain was synergistic with strain HD-551 subsp.kenyae(cry1A,cry2A, andcry1Bgenes) but not with HD-133 subsp.aizawai(cry1Ab,cry1B,cry1C, andcry1Dgenes) when the strains were cultured together in a cotton seed meal medium and fed toM. configurata. The growth rate, economic yield, and toxicity of the new isolate, A1.2/72, produced in a 14-L laboratory fermenter declined when the fermentation ingredients were tripled. We believe that the indigenous strain A1.2/72 warrants further research development for bertha armyworm control.

The effects of 6 fungicides (chlorothalonil, maneb, thiophanate-methyl, mancozeb, metalaxyl+mancozeb and zineb) and two herbicides (diquat and glufosinate-ammonium) used commercially in potato fields were evaluated in vitro on Beauveria bassiana (Balsamo) Vuillemin, a fungal pathogen of the Colorado potato beetle, Leptinotarsa decemlineata (Say). All 6 fungicides tested along with the herbicide glufosinate-ammonium inhibited B. bassiana mycelial growth and sporulation. However, the 2nd herbicide tested, diquat, had no noticable effect on B. bassiana mycelial growth and sporulation. Remarkably, diquat synergized the insecticidal activity of B. bassiana in the simultaneous treatments on Colorado potato beetle adults and caused 50–76.6% mortality.

The utilization profile of 49 carbohydrates, based on, API 50 CH biochemical tests, was used for the identification and the discrimination of 75 isolates of Beauveria and Tolypocladium . The data were used to construct a most parsimonious tree in which Beauveria and Tolypocladium were separated into two main branches. Although the Beauveria isolates could not be separated at the species level, eight different clusters corresponding to the eight Tolypocladium species studied were revealed. The reproducible carbohydrate utilization profiles of B. bassiana, B. brongniartii, T. cylindrosporum, T. niveum, T. geodes and T. tundrense have permitted the construction of an identification key that could serve as a tool to complement the morphological description of selected Beauveria and Tolypocladium isolates for their correct identification at the species level. The API 50 CH method is time-saving and can complement effectively more classical techniques for the discrimination between Beauveria and Tolypocladium and for the taxonomy of Tolypocladium at the species level.

Two Bacillus thuringiensis var. kurstaki HD-1 mutants, two Bacillus thuringiensis var. israelensis HD-500 mutants, and four rice grain dust isolates were characterized using microscopic examination and protein profiles of purified crystals on sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Specific detection of cryI- and cryIV-type genes was performed in a polymerase chain reaction using cryI and cryIV-specific oligonucleotide primers. The cry-type genes under study consisted of cryIA(a), cryI(A)b, cryI(A)c, cryIB, and cryIV. Presence or absence of the cryI- and cryIV-type genes was further confirmed by Southern blotting followed by hybridization with specific cryI and cryIV gene fragments. A genetically modified strain of B. thuringiensis var. kurstaki HD-1, called OZK-13 and obtained following mutagenesis with ozone, was shown to contain cryIA(a), cryIA(b), and cryIA(c) genes. Another kurstaki HD-1 mutant, called NGK-13 and obtained following treatment with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), was shown to have lost the cryIA(b) gene while retaining the cryIA(a) and cryIA(c) genes. NGI-23-1, an oligosporogenous–multicrystalliferous mutant of B. thuringiensis var. israelensis (Bti) HD-500, obtained following treatment with MNNG contained cryIV-type genes. NGI-22, an oligosporogenous–acrystalliferous mutant of Bti HD-500, contained no cryI- nor cryIV-type genes. The rice grain dust isolate BT-285 contained the cryIA(a) and cryIA(c) genes. Isolate BT-14 contained only the cryIA(c) gene, whereas isolate BT-209 contained cryIA(a), cryIA(b), and cryIB genes. Isolate BT-205 contained no cryI- nor cryIV-type genes. Bacillus thuringiensis mutants and natural isolates shown to contain cryI-type genes were tested for their insecticidal activities in a series of bioassays against Hyphantria cunea Drury (Lepidoptera: Arctiidae). All cryI-carrying strains were toxic against the insect larvae. BT-205 was also tested and exhibited no toxicity against the insect larvae.Key words: Bacillus thuringiensis, δ-endotoxin crystal, cry-type genes, polymerase chain reaction.

A simple and rapid assay for the detection of Bacillus weihenstephanensis isolates and other psychrotolerant strains in the Bacillus cereus group was developed. It is based on the presence of a nucleotide substitution at position 795 on the housekeeping pycA gene in all B. weihenstephanensis strains. This mutation creates a PstI recognition site. It is absent in mesophilic strains in the B. cereus group. The pycA gene is amplified by PCR and the amplicons submitted to PstI digestions. In mesophilic strains, a single band of 1,718 bp in length is visualised on an agarose gel. In B. weihenstephanensis strains and in all other psychrotolerant strains from the B. cereus group, the amplicons are cleaved and two bands of 1,175 and 543 bp, respectively, are visualised. This method could be used for the screening of B. cereus collections and for the identification of psychrotolerant and mesophilic isolates from different environments.

The Bacillus cereus group comprises seven bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus, and Bacillus weihenstephanensis. Bacillus weihenstephanensis is distinguished based on its capability to grow at 7 °C but not at 43 °C, and the presence of specific signature sequences in the 16S rRNA and cspA genes and in several housekeeping genes: glpF, gmK, purH, and tpi. Bacillus weihenstephanensis-specific signature sequences were found in some B. cereus and B. mycoides strains suggesting psychrotolerance. This was confirmed by growth at 7 °C but not at 43 °C. The other B. cereus and B. mycoides strains and all B. anthracis, B. thuringiensis, and B. pseudomycoides harbored the mesophilic signature sequences. The strains tested grew at 43 °C but did not grow at 7 °C. A maximum-likelihood phylogenetic tree was inferred from comparisons of the concatenated nucleotide sequences. Three groups and one branch were revealed. Group I, II, and III comprised the mesophilic B. cereus, some mesophilic B. mycoides, and all B. anthracis and B. thuringiensis strains; the psychrotolerant B. cereus and B. mycoides, and all B. weihenstephanensis strains; and some mesophilic B. mycoides and all B. pseudomycoides strains, respectively. The branch corresponds to the single B. cytotoxicus strain. Based on psychrotolerance and multilocus sequence analysis, further confirmed by comparisons of amino acid sequences, we show that some B. cereus and B. mycoides strains should be reclassified as B. weihenstephanensis.

The Bacillus cereus group sensu lato includes six closely-related bacterial species: Bacillus cereus, Bacillus anthracis, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides and Bacillus weihenstephanensis. B. thuringiensis is distinguished from the other species mainly by the appearance of an inclusion body upon sporulation. B. weihenstephanensis is distinguished based on its psychrotolerance and the presence of specific signature sequences in the 16S rRNA gene and cspA genes. A total of seven housekeeping genes (glpF, gmK, ilvD, pta, purH, pycA and tpi) from different B. thuringiensis serovars and B. weihenstephanensis strains were amplified and their nucleotide sequences determined. A maximum likelihood phylogenetic tree was inferred from comparisons of the concatenated sequences. B. thuringiensis serovars navarrensis, bolivia and vazensis clustered not with the other B. thuringiensis serovars but rather with the B. weihenstephanensis strains, indicative of a common phylogeny. In addition, specific signature sequences and single nucleotide polymorphisms common to B. thuringiensis serovars navarrensis, bolivia and vazensis and the B. weihenstephanensis strains, and absent in the other B. thuringiensis serovars, were identified.

Laboratory experiments were done to determine whether the adult Coleomegialla maculate lengi Timberlake was affected by ingesting M-One, a commercial formulation of Bacillus thuringiensis var. san diego used to control the Colorado potato beetle, Leptinotarsa decemlineala (Say). Over a period of 10 consecutive days, consumption of pollen contaminated with 200 ml M-One per liter (5.6 × 109 CPBIU per liter) had no lethal effects on adult C. maculata. The level of predation by adult C. maculata on Colorado potato beetle eggs was reduced by M-One. At the manufacturer’s recommended field rate of 20 ml M-One per liter (i.e., 5.6 × 108 CPBIU per liter), the number of eggs attacked was 37.0 and 14.5% lower than the untreated eggs, respectively, 3 and 24 h after the beginning of the test. The feeding behavior and results of a water versus 200 ml M-One per liter two-choice test suggest that the decrease in food intake caused by M-One cannot be explained by a α-endotoxin-induced paralysis of the mouth parts or midgut.

We have designed a simple and rapid assay for chloroplast-based triazine resistance in higher plants using PCR amplification of thepsbA gene coupled toMaeI digestion of the amplified product to distinguish triazine resistant from sensitive biotypes. Our assay is universal and avoids the need of lengthy procedures of previously published assays, which either required spraying of seedlings in a controlled environment, quantification of chlorophyll fluorescence of leaf discs after incubation in triazine solution, DNA sequencing of thepsbA gene, or Southern-blot analysis. Our diagnostic system is qualitative, reliable, fast and simple. More than 100 seedlings taken directly from the field can be analyzed in one day. This system has a direct application towards a more rational use of herbicides in production fields. It also represents a valuable tool to monitor spreading of resistant biotypes through time and space and can serve as a model system applicable to other gene monitoring needs.

The Bacillus cereus sensu lato group comprises six related species: B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis. Bacillus thuringiensis is a mesophile. It is distinguished from other members of the B. cereus group by the apparition of an inclusion body upon sporulation. B. weihenstephanensis, however, is a psychrotolerant. It grows at 7 degrees C but not at 43 degrees C. It is further characterised by the presence of specific signature sequences on two genes, the 16S rRNA gene (the small subunit ribosomal RNA gene) and cspA (encoding the major cold shock protein). Five B. thuringiensis serovars selected from previous studies, bolivia, vazensis, navarrensis, azorensis and asturiensis were studied here for their capability to grow at 7 degrees C but not at 43 degrees C. Next, their 16S rRNA and cspA genes were analysed for the presence of B. weihenstephanensis-specific signature sequences. Bacillus thuringiensis serovars bolivia, vazensis and navarrensis met the description of B. weihenstephanensis.

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar.

In Bacillus thuringiensis , the hag gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Specific flagellin amino acid sequences have been correlated to specific B. thuringiensis H serotypes, H1 to H67. Ten H serotypes, however, contain three or more antigenic subfactors, labeled a, b, c, d, or e, and have been subdivided into 23 serovars. In the present study, we set out to analyze the sequence diversity of flagellins among serovars from the same H serotypes. We studied the hag genes in 39 B. thuringiensis strains representing the 23 serovars from the 10 H serotypes mentioned above. A serovar and a biovar from an 11th H serotype were also included. The hag genes were amplified and cloned and their nucleotide sequences were determined and translated into amino acid sequences, or the sequences were retrieved directly from GenBank when available. Strains of the H3 serotype contained two or three copies of the fla gene, an ortholog of the hag gene. Strains of the H6 serotype contained three copies. Strains of all other H serotypes each contained a single copy of the hag gene. Alignments of amino acid sequences from all copies in all strains of the H3 serotype revealed short signature sequences, GGAG and SGG, GPDPDDAVKNLT, and DITTTK, that appeared to be specific to the H3c, H3d, and H3e antigenic subfactors, respectively. Similar short signature sequences, GDIT, AFIK, TSAGKA, and SAPSKG, were revealed for H8b, H8c, H20b, and H20c, respectively. Amino acid sequences in the flagellin central variable region were highly conserved among serovars of the H3, H5, H11, and H20 serotypes and much more divergent among serovars of the H4, H10, H18, H24, and H28 serotypes. Two bootstrapped neighbor-joining trees were respectively generated from the alignments of the amino acid sequences translated from all copies of the hag genes in the B. thuringiensis strains of the H3 and H6 serotypes. Sequence identities and relationships were revealed. A third bootstrapped neighbor-joining tree was generated, this one from the alignment of the flagellin amino acid sequences from all the B. thuringiensis strains in the study. Eight clusters, I to VIII, were revealed. Although most clusters contained strains and serovars from the same H serotype, clusters VII and VIII contained serovars from different H serotypes.

A 5.7-kb EcoRI fragment was cloned from plasmid DNA of Bacillus thuringiensis strain M15. It contains two insertion sequences (IS), IS231M2 and -M1 in the 5'-3' order, arranged in tandem, in same orientation, separated by a 540-bp region. The primary structure is typical of a composite transposon, here of 3847 bp in length, for which the name Tn231M is proposed. Each IS is delimited by 18-bp inverted repeats (IR), and flanked by 11-bp direct repeats (DR). Both IS share 99.3% nucleotide identities. IS231M1 has a single open reading frame (ORF) which encodes a putative 477-amino-acid transposase. IS231M2 has two smaller ORFs: ORF1 and ORF2, which could code for polypeptides of 329 and 118 amino acids in length, respectively. Further analysis reveals that the regions upstream of IS231M2, and downstream of -M1, and the 540-bp region, contain additional pairs of IR and DR. Interestingly, potential annealing between all pairs of IR and DR could generate two unusual cloverleaf secondary structures.

In Escherichia coli, the fliC gene encodes flagellin, the protein responsible for eliciting the immunological reaction in H serotyping. Here, the presence of the flagellin fliC gene was studied in 86 Bacillus thuringiensis strains encompassing 67 H serotypes. Nineteen strains from four additional species in the B. cereus sensu lato group, B. cereus, B. anthracis, B. mycoides, and B. weihenstephanensis, were added for comparison purposes. The fliC genes were amplified, cloned and their nucleotide sequences determined and translated into amino acid sequences. A bootstrapped neighbor-joining tree was generated from the alignment of the translated amino acid sequences of the amplicons. Although most B. thuringiensis H serotypes had different flagellin amino acid sequences, some different B. thuringiensis serovars shared identical flagellin amino acid sequences. In addition, although serovars from the same H serotype were sometimes found clustered together, several serovars from the same H serotype carried flagellins with sufficiently different amino acid sequences as to be located on distant clusters. No correlations could be established between flagellin (FliC) protein sequence diversity among B. thuringiensis H serotypes and H serotype diversity. These suggest that the B. thuringiensis fliC gene does not code for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. In a previous study, the authors have shown that the B. thuringiensis hag gene codes for the flagellin copy responsible for eliciting the immunological reaction in H serotyping. It is proposed that the B. thuringiensis fliC gene studied here be renamed and that the so-called hag gene studied before be renamed fliC, both in accordance with the E. coli nomenclature.

Lygus hesperus Knight (Hemiptera: Miridae) is an economically important insect pest controlled primarily by chemical pesticides. Bacillus thuringiensis Berliner is a gram-positive bacterium, which upon sporulation produces a parasporal inclusion body, the crystal. The latter, in some strains, exhibits specific insecticidal activities against lepidopteran, coleopteran, or dipteran pests. The aim of the present work was to develop a Bacillus thuringiensis-based assay on L. hesperus. Several factors involved in the expression of the B. thuringiensis insecticidal activity were assayed for their effects on L. hesperus mortality. We show that the choice of the L. hesperus diet type, the L. hesperus developmental stages, the B. thuringiensis crystal alkaline solubilization buffers, the dialysis buffers, the dialyzed solubilization buffers, and the proteolytic activation can all significantly affect the L. hesperus viability. This work provides essential information in pinpointing key steps before the establishment of a thorough screening program for B. thuringiensis strains expressing antihemipteran activity.

The distribution of IS231 has been analyzed in Bacillus thuringiensis serovars. A 723-bp HaeII conserved fragment from IS231M has been used as a probe against EcoRI-digested B. thuringiensis total DNA to yield serovar-specific hybridization profiles. The approach was useful at revealing the extent of distribution of IS231-like sequences between and within strains. Of the 88 B. thuringiensis strains tested, 70 showed hybridization banding patterns that comprised between one and 20 distinct bands. These 70 B. thuringiensis strains were grouped based on banding pattern similarities. Interestingly, intraserovar strains did not necessarily cluster together.