Density and depth variations of Daphnia multilocus genotypes during a summer period in Lake Maarsseveen

The genotype composition of a Daphnia population complex during a summer period in Lake Maarsseveen (The Netherlands) was determined by allozyme analysis. The depth distribution, diel vertical migration and several parameters of the total population were measured. Young-of-the-year (0⁺) perch (Perca fluviatilis) were caught and species and allozyme types of Daphnia in the perch gut were also analysed. During May 1997, the densities of D. hyalina, D. galeata, the back-cross D. g × h — hyalina and the multilocus allozyme genotypes of the hybrid D. g × h decreased, except one multilocus genotype (MMMF). Total population size decreased and the ratio of females with eggs to those without eggs decreased as well. Food limitation during this clear-water phase in the lake is considered responsible. All genotypes, except MMMF, gradually descended in the water column. This drift is thought to be a reaction to the abundantly present 0⁺ perch or to the kairomones of this fish, although predation on the daphnids was still absent. In June, diel vertical migration started, except again part of the MMMF subpopulation. The other part migrated over a short distance compared with the other taxa and allozyme types. Within two weeks, the upper 5 m of the epilimnion was devoid of Daphnia, and guts of perch were predominantly filled with MMMF. The daphnids in the gut and the lake did not differ in allozyme type composition. By the end of July, population density had increased again. The size and composition of the Daphnia population complex continuously changed during the study period, as did the depth distribution of the components. Different genotypes within the population complex seem to have developed different strategies to cope with starvation and predation and the state at a particular moment can be understood only if past and present factors are considered.     

Determination of diarrhetic shellfish toxins in various dinoflagellate species

Sixteen species of unialgal samples of dinoflagellate, either wild or cultured, were tested for production of diarrhetic shellfish toxins such as okadaic acid (OA), dinophysistoxin-1 (DTX1), and pectenotoxins (PTXs). Determination of micro-quantities of the toxins was facilitated by fluorometry and UV HPLC. Seven Dinophysis species were confirmed to produce either OA or DTX1, or both. Toxin content and composition varied regionally and seasonally. Intraspecies variation was also observed among cultured strains of Prorocentrum lima. PTX2 was the only toxin detected among PTX family, and D. fortii was the only species to contain this toxin. author for correspondence     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

Anatoxin-a concentration inAnabaena andAphanizomenon under different environmental conditions and comparison of growth by toxic and non-toxicAnabaena-strains — a laboratory study

Anatoxin-a-concentration in cells ofAnabaena- andAphanizomenon-strains and in their growth media were studied in the laboratory in batch cultures at different temperatures, light fluxes, orthophosphate and nitrate concentrations and with different nitrogen sources for growth. Toxin concentrations were detected by HPLC. Also, the growth of the toxicAnabaena-strains was compared to that of a non-toxic one. The non-toxicAnabaena was never found to produce anatoxin-a. The amount of toxin in the cells of the toxic strains was high, often exceeding 1% of their dry weight. High temperature decreased the amount of the toxin regardless of growth. Growth limiting low and growth inhibiting high light decreased the amount of the toxin in the cells ofAnabaena-strains. The highest light flux studied did not limit the growth or decrease the level of the toxin in the cells ofAphanizomenon. Growth in N-free medium (i.e. N₂ fixation) showed that the cells contained more toxin than growth in N-rich medium. Orthophosphate concentration had no effect on toxin levels, although the lowest concentrations limited the growth of all strains studied. The toxic strains tolerated higher temperatures than the non-toxic one, but the non-toxic strain seemed to be more adjustable to high irradiance than the toxic ones. The yields (dry weight) of non-toxic and toxic strains differed significantly in different phosphate concentrations.     

Erythrogenic toxins A, B and C: Occurrence of the genes and exotoxin formation from clinical Streptococcus pyogenes strains associated with streptococcal toxic shock-like syndrome

We report the study of 53 clinical isolates of group A streptococci, all from patients with streptococcal toxic shock-like syndrome. The strains were analysed for the occurrence of the genes of erythrogenic toxins (pyrogenic exotoxins) types A, B and C and in vitro production of these toxins. In contrast to reports indicating that 85% of the toxic shock-like syndrome-associated isolates contained the erythrogenic toxin A gene, only 58.5% of our strains harboured this gene. The erythrogenic toxin C gene was detected in 22.6% of the isolates. Erythrogenic toxin A and erythrogenic toxin B were produced by 68.7% and 58.3% of the strains containing either gene. For all group A streptococci, irrespective of clinical association, the erythrogenic toxin B gene was detected in all the isolates tested. Thus, it is difficult to define a specific role for erythrogenic toxin B in toxic shock-like syndrome as there was no clear correlation between this disease and the presence of toxin genes. Our results suggest the existence of other pathogenic factor(s) produced by group A streptococci which may stimulate human peripheral T lymphocytes in a manner similar to that of erythrogenic toxins, thus explaining different observations in previous epidemiological genetic studies.     

A type II restriction endonuclease of Streptococcus thermophilus ST117

Three separate sets of polymerase chain reaction primers were designed to specifically detect the presence of a toxin A gene fragment, a toxin B gene fragment, and the entire toxin B gene. In addition toxin gene fragments that were amplified from well characterized toxic strains were tagged fluorescently and used as hybridization probes to screen C. difficile strains. A survey of 37 toxic strains and 10 non-toxic strains demonstrated that toxic strains normally contain the genetic composition for toxin A and toxin B simultaneously; whereas, non-toxic strains typically did not contain detectable toxin determinants. The only exception found was strain 39, which had the genetic composition for toxins A and B, but was not cytotoxic under the conditions tested.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Phenotypic and genetic features of cultural-morphologic variants of Bacillus anthracis

Comparative analysis of MVLA-genotypes of 6 Bacillus anthracis strains and 40 their variants differing on capsule- and toxin synthesis, hemolytic, proteolytic and lecitinase activity, nutritional requirements, susceptibility to anthrax bacteriophages, virulence, immunogenicity, and presence of genes for capsule and toxin synthesis was performed. Results of phylogenetic analysis of 5 chromosome locuses and plasmid locus pXO1aat which are variable for this sample of B. anthracis cultures showed that all strains divided on 2 main clusters - A and B. Cluster A consisted of 5 genotypes whereas cluster B - of 1 genotype. All highly virulent original strains and variants with characteristic phenotype Cap(CO2)(+)(O2)(-)Tox(+)ProtA(+)Hly(+) Lec(-)Trp(+) had identical genotype in 4 groups and in 5th group differences were present only in vrrA locus. All original strains and variants with the most atypical complex of phenotypic characteristics Cap (CO2)(+)(O2)(+)Tox(-)ProtA(-)Hly(-)Lec(-)Trp(-) also had the same genotype belonging to cluster B and diverged on characteristic of 5 chromosomal VNTR locuses and pXO1aat locus from typical strains. Absence of toxin production in vitro was not related to loss of genetic determinants of toxin components. Cultures with typical characteristics, one of which was ability to produce toxin in vitro, had larger sizes of amplicons of pXO1aat locus (135 and 132 nbp), whereas atoxigenic original strains and variants with complex of atypical characteristics and identical chromosome genotype had the smallest sizes (123 bnp). All original cultures were isolated in Russia, their genotypes are described for the first time.     

Kinetics of plasmid transfer among <Emphasis Type="Italic">Bacillus cereus </Emphasis>group strains within lepidopteran larvae

The cry toxin encoding plasmid pHT73 was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to six B. cereus group strains in three lepidopteran (Spodoptera exigua, Plutella xyllostella and Helicoverpa armigera) larvae by conjugation. The conjugation kinetics of the plasmid was precisely studied during the larval infection using a new protocol. The infections were performed with both vegetative and sporulated strains. However, larval death only occurred when infections were made with spore and toxin preparations. Likewise, spore germinations of both donor and recipient strains were only observed in killed larvae, 44–56 h post-infection. Accordingly, kinetics showed that gene transfer between B. thuringiensis strain KT0 and other B. cereus strains only took place in dead larvae among vegetatively growing bacteria. The conjugational transfer ratios varied among different strain combinations and different larvae. The highest transfer ratio reached 5.83 × 10⁻⁶ CFU/donor between the KT0 and the AW05R recipient in Helicoverpa armigera, and all transconjugants gained the ability to produce the insecticidal crystal. These results indicated that horizontal gene transfer among B. cereus group strains might play a key role for the acquisition of extra plasmids and evolution of these strains in toxin susceptible insect larvae.     

Purification and biochemical characterization of a basic superantigen (SPEX/SMEZ3) from Streptococcus pyogenes

A potent basic superantigen (designated streptococcal pyrogenic exotoxin X, SPEX/SMEZ3) was purified to homogeneity from culture supernatants of a Streptococcus pyogenes scarlatina strain of type 12 (genotype speA(-), speC(-)) and characterized. Sequence alignments revealed SPEX to be an allele of the streptococcal mitogens type Z (SMEZ). The N-terminal amino acid sequence of SPEX was found with LEVDNNSLLR to be identical to the recently described acidic superantigen SMEZ. Although SPEX/SMEZ genes were present in all of the streptococcal strains tested, a toxin production could only be detected in a small number of strains. The produced toxin concentration in the culture supernatants of positive strains differed between 0 and 20 ng ml(-1). The purified SPEX stimulated human T-lymphocytes with Vbeta8 specificity at extremely low concentrations (lower than 100 pg ml(-1)).     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

An experimental analysis of the relationships between fitness components and malic enzyme genotypes inTribolium confusum

The hypothesis that natural selection is capable of maintaining allozyme variation in natural populations was tested using a species of flour beetles,Tribolium confusum. We selected a polymorphic locus (a locus encoding variation for malic enzyme) in an experimental population ofT. confusum and scored the genotypes at this locus for a series of fitness components on different flour types. Measurements included survival rate, development time, fecundity, and rate of egg cannibalism. Flour type had significant effects on most traits. Significant differences among genotypes for fecundity and rates of egg cannibalism and the presence of genotype × flour type interactions for development time were demonstrated. Thus, changes in allele frequencies at the malic enzyme locus could in part be under the influence of natural selection. The existence of genotype × flour type interactions suggests that environmental heterogeneity could maintain allozyme variation at the malic enzyme locus.     

Comparison of AP-PCR methods,ribotyping (PCR-ribotyping) and pulsed-field electrophoresis (PFGE) for strains of Clostridium difficile producing toxin B and not producing toxin A

In this study were used AP-PCR, PCR-ribotyping and pulsed-field elecrophoresis (PFGE) for comparative study of toxin A-negative/toxin B-posi-tive Clostridium difficile strains with deletion in toxin A gen. We investigated nine unrelated clinical strains, isolated from different units and different time from patients suffering to antibiotic associated diarrhea (AAD). We found that toxin A-negative/toxin B-positive C. difficile strains isolated in Poland belonging to a single genotype A, are being similar to the Japanese strains.     

Morphological and cytogenetic characteristics of intergroup hybrids between closely related mating groups of the Closterium ehrenbergii species complex (Desmidiales, Chlorophyta)

Five F₁ hybrid strains were established from rare survivors in intergroup crosses between three closely related mating groups (A, B and H) of the Closterium ehrenbergii Meneghini ex Ralfs species complex. Cell sizes of these five strains studied under our standard culture conditions were compared to those of their parental stains and also to the total range of cell-size variation in each mating group. All five F₁ strains were larger in mean cell width than their parental strains. In cell length, three of them were larger than, one was the same as, and the other was intermediate between their parental strains. Their cell sizes were always larger than the range of their respective smaller parental mating group and three of them were larger than the range of their respective larger parental mating group.     

Further evidence of thermostability variation within electrophoretic mobility classes of enzymes

Two Drosophila melanogaster strains, each heterozygous for fast and slow alleles at the Adh locus, and each having balanced second chromosomes, were found to differ in the apparent thermostability of the slow allozyme. The two strains were crossed, and F₁heterozygotes were separated on the basis of the origin of the slow allele. After electrophoresis, the cellulose acetate strips were treated 1 1/2 min at 35 C. The putatively more sensitive allozyme showed a strikingly greater response to heat. These findings further support the conclusion that electrophoretically cryptic allelic differences exist which are expressed in thermostability differences. Further application of this approach has revealed one similar sensitive slow allozyme and three cases of a relatively resistant fast ADH allozyme in wild-caught flies.This work was supported by NIH Grant GM18967-02-03.     

Evaluation of different methods for detection of Clostridium difficile toxins in Poland

The aim of this study was to compare different methods for C. difficile toxins detection. Fifty three stool samples taken from patients with antibiotic-associated diarrhoea were studied. TCD toxin A EIA (Becton Dickinson, USA), Tox A/B ELISA test (TechLab, USA), cytotoxicity and neutralization assay on McCoy cells and PCR for detection of both toxin A and B genes were performed in vivo (in stool samples) and in vitro (in isolated strains). Reference toxigenic and nontoxigenic and two Japanese toxin A-negative and toxin B-positive C. difficile strains were used as a controls. TCD toxin A EIA detected in vivo only 19 positive samples. Tox A/B test detected 52 positive samples out of 53 studied. All 53 stool samples were C. difficile culture positive (53 strains were cultured). Toxin B was detected in 52 strain-supernatants and in all controls (except the nontoxigenic one). Both toxin A and B genes were detected by PCR in all 53 isolated strains, Japanese and reference strain (except the nontoxigenic one). In vitro toxin A was detected by TCD toxin A EIA in 42 strains. These results were compared with those obtained in Tox A/B ELISA test. We observed 52 positive strains. Toxigenic reference strain and two Japanese toxA(-)/toxB(+) strains were also positive. Only 2 negative results were obtained with the nontoxigenic reference strain and unique nontoxigenic isolated strain. Tox A/B ELISA test seems to be the best for detection of C. difficile toxins in vivo and in vitro. Test avoids the false-negative results in the case of presence of toxin A-negative and toxin B-positive strain.     

Killer toxin producing strains of the yeasts Hanseniaspora uvarum and Pichia kluyveri

By heat treatment killer strains of the type K1 of Saccharomyces cerevisiae that are known to harbour dsRNA plasmids were completely cured, whereas only a small fraction of the clones of the killer type K2 had lost the dsRNA dependent killer character. The K2 killers but not the strains of killer type K1 were easily cured by cycloheximide. Killer strains of Hanseniaspora uvarum were not curable by heat treatment. Curing was successfull with cycloheximide or 5-fluorouracil. Two double-stranded RNA plasmids were detected in the killer strains of H. uvarum. The smaller dsRNA plasmid was absent in the strains that were cured of their killer character by 5-fluorouracil. The killer character of H. uvarum was transferred to S. cerevisiae by spheroplast fusion. The fusion products showing the killer character contained both dsRNA plasmids, obviously the smaller plasmid (M-dsRNA) carries the genes for killer toxin formation. Killer strains of Pichia kluyveri were not curable of their killer character, in these strains no dsRNA plasmids were detected.This paper was kindly supported by a grant from the Deutsche Forschungsgemeinschaft