Differentiation of capripoxvirus species and strains by polymerase chain reaction

A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine stains used in Russia and countries of the former Soviet Union from epizootic isolates of sheep pox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.     

Differentiation of capripoxvirus species and strains by polymerase chain reaction

A fast and simple method for capripoxvirus species identification has been developed. The method is based on multiplex polymerase chain reaction (MPCR) with species-specific primers and does not require nucleotide sequencing or restriction analysis of PCR products. To differentiate vaccine strains used in Russia and countries of the former Soviet Union from epizootic isolates of sheeppox virus, a method based on restriction analysis of the ankyrin-repeat protein gene fragment amplified by PCR has been developed. Being highly specific, both methods may be used for routine diagnosis of capripoxvirus-associated diseases.     

Platelet aggregation induced by strains of various species of coagulase-negative staphylococci

Major species of coagulase-negative staphylococci (CNS) were tested for their ability to induce platelet aggregation in rabbit platelet-rich plasma (PRP). Among 11 species of CNS tested, a majority of the strains of 10 species of CNS (S. epidermidis, S. simulans, S. capitis, S. hyicus, S. sciuri, S. cohnii, S. xylosus, S. hominis, S. haemolyticus, S. warneri) caused induction of the platelet aggregation and serotonin release, while S. saprophyticus did not show such activity. The addition of aspirin (10 mM) or quinacrine (1 mM) to PRP resulted in no remarkable effect on the platelet aggregation induced by these strains and it was shown that the platelet aggregation did not require arachidonate pathways. Complement system components were shown to be one of the plasma factors required for platelet aggregation by ten strains of each species of CNS. The bacterial substance participating in the platelet aggregation by ten species of CNS tested was indicated to be heat-stable and trypsin-resistant, while the activity of a strain of S. epidermidis was susceptible to trypsin.     

Two species ofMucor with oval- and spherical-spored strains

Mucor racemosus Fres. andM. sphaerosporus Hagem have several morphological features in common; intermediate forms are known and matings of typical representatives of both species produce abundant zygospores. For these reasonsM. racemosus andM. sphaerosporus are accepted as an oval-spored and a spherical-spored form of one species:M. racemosus f.racemosus andM. racemosus f.sphaerosporus.The same applies toM. griseocyanus Hagem andM. janssenii Lendner, and the namesM. griseocyanus f.griseocyanus andM. griseocyanus f.janssenii are proposed.     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Diversity and affinities among species and strains of Lipomyces

Phylogenetic relationships of the yeast genus Lipomyces were studied using sequences from fragments of 5.8S rRNA gene and from internal transcribed spacer region ITS2 of 13 strains (7 type strains included) representing five species and subtaxa, and originating from different geographical locations (Japan, Trinidad, Nigeria, North America, Western Europe, Russia, South Africa, Mauritius). Parsimony and distance analyses were performed. Tree topology from the parsimony and distance analyses of the sequences confirmed the results of nDNA reassociation. Results segregate the 13 isolates of Lipomyces into five major clades.     

Contamination by uranium mine drainages affects fungal growth and interactions between fungal species and strains

The presence of aquatic hyphomycetes has been reported for several heavy metal-contaminated waters. Tolerance probably is one adaptation to coping with heavy metals. To help clarify this issue strains of two species of aquatic hyphomycetes (Tricladium splendens Ingold and Varicosporium elodeae Kegel) were isolated from a reference stream and a stream contaminated with heavy metals and grown on malt extract agar prepared with reference and contaminated water to characterize colony morphology, growth rate, growth inhibition and interaction among species and strains. In V. elodeae the morphology of colonies differed between strains. Colony diameter increased linearly over time with growth rates being lower for strains isolated from contaminated than from reference streams (mostly for V. elodeae). Strains from the contaminated stream grew faster in medium prepared with contaminated water than in medium prepared with reference water, while for strains from the reference stream there was no significant difference in growth rates on the two media. In interacting isolates radial growth toward the opposing colony was generally lower than toward the dish edge. Percentage growth inhibition was higher for isolates in intraspecific interactions (13-37%) than in interspecific interactions (3-27%). However differences in growth inhibition experienced by interacting isolates were observed only in three cases out of 16. The difference between the percentage inhibition caused and experienced by a given isolate was highest in interactions involving isolates with distinct growth rates. Our results suggest that strains from the reference stream tolerate heavy metals while strains from the contaminated stream seem to be adapted to contaminated waters. We hypothesize that in natural environments fungal species-specific limits of tolerance to metal contamination might determine an abrupt or gradual response of the original fungal community to mine pollution giving origin to a poorer fungal community dominated by adapted strains with distinct functional efficiency.     

Effect of different west african species and strains ofArthrobotrys nematophagous fungi onMeloidogyne species

Different species ofArthrobotrys nematophagous fungi and several strains ofA. oligospora have been studied for their antagonistic effects against nematodes of the genusMeloidogyne, important pests of vegetables. All fungi trappedM. mayaguensis andM. incognita juvenilesin vitro but had no effect on the juveniles ofM. javanica. In pot experiments withM. mayaguensis, all fungi reduced the nematode populations and stimulated the growth of tomato seedlings. In a field trial, a strain of A.oligospora, isolated in Senegal and incorporated into compost blocks, was efficient in increasing the tomato seedling growth. The introduction of nematophagous fungi in compost blocks as a biological biocontrol technique against phytophagous nematodes adapted for developing countries is discussed.     

Leishmania species: mechanisms of complement activation by five strains of promastigotes

The interaction of fresh serum with promastigotes of Leishmania major, L. donovani, L. mexicana mexicana, L. mexicana amazonensis, and L. braziliensis guyanensis results in lysis of all strains tested with either fresh human or guinea pig serum at 37 C for 30 min. Lysis does not occur in the cold and requires divalent cations and complement that is active hemolytically. Serum deficient in the eighth component of complement is not lytic. Lysis of L. major, L. mexicana, and L. braziliensis proceeds fully in human serum containing EGTA/Mg2+ or in guinea pig serum deficient in the fourth complement component. These species consume only small amounts of C4 from human serum and do not require calcium to optimally bind C3. The data indicate that all are activators of the alternative complement pathway and that the classical pathway is not required for the lysis of these organisms. Promastigotes of L. donovani, in contrast, activate the classical pathway. The presence of calcium is required for both optimal C3 binding and parasite lysis, and L. donovani promastigotes consume C4 when incubated in human serum. In high concentrations, human serum agglutinates all tested Leishmania spp. The agglutinating factor does not require divalent cations, is heat stable, and works at 4 C, suggesting that it is an antibody. This "naturally occurring" antibody cross reacts with all Leishmania spp. and agglutinates them. The adsorption of serum with any Leishmania species or with beads that are Protein A coated, removes the agglutinogen. This factor causes a slight enhancement in alternative pathway activation by L. major and mediates the classical activation by L. donovani. In adsorbed serum, L. donovani promastigotes only weakly activate the alternative complement pathway. Increased concentrations of adsorbed serum are therefore necessary for lysis to proceed. The titer can be partially restored by the addition of heat inactivated serum. Using purified components of the classical cascade, we are unable to visualize surface bound C3 on L. donovani promastigotes unless heat inactivated serum is also present. We conclude that all Leishmania spp. promastigotes are susceptible to lysis by normal serum independent of antibody. The presence of small amounts of naturally occurring antibody in human serum enhances the susceptibility of L. donovani promastigotes to lysis by activating the classical complement pathway.     

Rate of trypanosome killing by lectins in midguts of different species and strains of Glossina

The activity of lectins in different species of tsetse was compared in vivo by the time taken to remove all trypanosomes from the midgut following an infective feed and in vitro by agglutination tests. Teneral male Glossina pallidipes Austen, G. austeni Newstead and G. p. palpalis R-D. removed 50% of all Trypanosoma brucei rhodesiense Stephens & Fantham infections within 60 h. A 'refractory' line of G. m. morsitans Westwood took 170 h to kill 50% infections while a 'susceptible' line of the same species failed to kill 50%. Agglutination tests with midgut homogenates showed differences between fly stocks which accorded with differences in rate of trypanosome killing in vivo. Flies fed before an infective feed were able to remove trypanosomes from their midguts more quickly than flies infected as tenerals. Increasing the period of starvation before infection increased the susceptibility to trypanosome infection of non-teneral flies. Teneral flies showed little agglutinating activity in vitro, suggesting that lectin is produced in response to the bloodmeal. Feeding flies before infection also abolished the differences in rate of trypanosome killing found between teneral 'susceptible' and 'refractory' G. m. morsitans, suggesting that maternally inherited susceptibility to trypanosome infection is a phenomenon limited to teneral flies. Electron micrographs of midguts of G. m. morsitans suggest that procyclic trypanosomes are killed by cell lysis, presumably the result of membrane damage caused by lectin action.     

Mutation and reversion frequencies of different Sulfolobus species and strains

We have determined the apparent and actual spontaneous mutation frequencies and rates for different species and strains of the thermoacidophilic crenarchaeote Sulfolobus. The proportion of mutations caused by insertion sequences has also been analyzed. Mutation frequencies for S. islandicus (0.08–0.6 mutations per cell division and 10⁷ cells) were below those determined for S. solfataricus and comparable to or lower than those for S. acidocaldarius. The proportion of insertion sequence mutations for the S. islandicus strains REN1H1 (9 out of 230) and HVE10/4 (0 out of 24) was found to be considerably lower than in S. solfataricus P1 and P2 and also low in comparison to other S. islandicus strains. Mutants defective in either the pyrEF genes or the lacS gene have been isolated. Their growth phenotype on selective and non-selective medium was examined and the inactivating mutations in either of the genes were determined. In addition the reversion frequencies for these mutants were measured and found to be in the range of <0.6–1.5 mutations per cell division and 10⁸ cells. However, when being subjected to electroporation as a transformation procedure, increased reversion was observed.     

Analysis of DNA from various species and strains of malaria parasites by restriction endonuclease fingerprinting

1. DNA from various rodent Plasmodium species and strains and from P. falciparum, the human parasite, were analysed by agarose gel electrophoresis following digestion with restriction endonucleases EcoRI, Hind III and Bam Hl. Complex patterns of ethidium-stained bands were obtained, which showed similarity but reproducible differences among the various parasite species (P. chabaudi, P. yoelii, P. berghei and P. falciparum). 2. No differences could be discerned among two cloned strains of P. yoelii (33X, and YM) and among pyrimethamine-resistant (pyrimethamine + chloroquine)-resistant and the drug-sensitive P. chabaudi clone from which the resistant clones were derived. 3. From the known complexity of Plasmodium DNA it could be concluded that the visible bands were derived from repetitive DNA fractions.     

Molecular relationships between closely related strains and species of nematodes

Summary Electrophoretic comparisons have been made for 24 enzymes in theBergerac andBristol strains ofCaenorhabditis elegans and the related species,Caenorhabditis briggsae. No variation was detected between the two strains ofC. elegans. In contrast, the two species,C. elegans andC. briggsae exhibited electrophoretic differences in 22 of 24 enzymes. A consensus 5S rRNA sequence was determined forC. elegans and found to be identical to that fromC. briggsae. By analogy with other species with relatively well established fossil records it can be inferred that the time of divergence between the two nematode species is probably in the tens of millions of years.The limited anatomical evolution during a time period in which proteins undergo extensive changes supports the hypothesis that anatomical evolution is not dependent on overall protein changes.     

Bacteroides species: maintenance of laboratory strains.

A medium composed of blood agar base (40 g/liter), yeast extract (5 g/liter), and cysteine hydrochloride (0.05 g/liter), completely filling screw-cap tubes (13 by 100 mm), can keep Bacteroides species alive for at least 10 months without refrigeration.     

Characterization of mannitol producing strains of Leuconostoc species

Mannitol is a naturally occurring low calorie sweetener, widely used in the food, pharmaceutical, medicine and chemical industries. In this study mannitol producing strains of Leuconostoc spp. (210) were isolated from a wide array of sources such as raw milk, fermented milks, fermented cereal foods, fruits, vegetables and sugar factory syrup. During initial screening, half of the population of these isolates (105) exhibited ability to produce mannitol to a variable extent. Only 11.4% isolate produced mannitol yield of above 80% (when fructose used @ 50 g/l). Cultural and environmental factors affecting growth and mannitol production were studied for four high mannitol producing isolates. High mannitol production was favored by high temperature and high pH. Isolates had high osmotic tolerance as these could use fructose concentration as high as 100 g/l in batch culture. Sequencing of 16S rRNA genes of the strains revealed that Ln27, Ln104 and Ln206 were Leuconostoc mesenteroides and Ln92 was Leuconostoc fallax.     

Plasmid profile of strains of various Legionella species

The plasmid profile of Legionella strains of different origin has been studied. 15 out of 32 Legionella cultures belonging to different strains have been found to contain plasmid DNA in an amount of 1 or 2 plasmids, with the exception of L. feelei having 6 plasmids. Only 1 out of 3 Legionella strains isolated in the USSR has been found to possess a plasmid with a molecular weight of about 80 MD. Plasmids with this molecular weight have been found in 13 Legionella strains under study, such plasmids in L. pneumophila strains of serogroup 1 (strains Flint 1 and Albuquerque 1) and serogroup 9 (strain No. 35282) having an exact molecular weight of 82.4 +/- 2.4 MD and being similar in molecular structure, which has been established as the result of their treatment with restrictases Pst 1 and Hind III.     

Assigning strains to bacterial species via the internet

Background  

Methods for assigning strains to bacterial species are cumbersome and no longer fit for purpose. The concatenated sequences of multiple house-keeping genes have been shown to be able to define and circumscribe bacterial species as sequence clusters. The advantage of this approach (multilocus sequence analysis; MLSA) is that, for any group of related species, a strain database can be produced and combined with software that allows query strains to be assigned to species via the internet. As an exemplar of this approach, we have studied a group of species, the viridans streptococci, which are very difficult to assign to species using standard taxonomic procedures, and have developed a website that allows species assignment via the internet.