Intraspecies genotypic heterogeneity among Mycoplasma gallisepticum strains

The DNA cleavage patterns and protein profiles of six Mycoplasma gallisepticum strains from various parts of the world were compared. Obvious differences among the strains were obtained by DNA restriction analysis. Reflection of genotypic variations in the polypeptide patterns was less pronounced; slight differences in the protein profiles of the strains were found. The data presented here indicate that some intraspecies polymorphism exists among M. gallisepticum strains.     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

Molecular biological differences between strains of Mycoplasma gallisepticum]

Differences in virulence of two Mycoplasma gallisepticum strains, S6 and A5969, are confirmed in experiments with chickens. Macromolecular discrepancies detected between these two strains are concerning the genomic size, electrophoretic spectra of DNA and proteins. Cross immunoblotting data with polyclonal and monoclonal antibodies reveal major immunogens of protein nature in both the strains. Homologous proteins with different electrophoretic mobility are detected in other four M. gallisepticum strains. A possible participation of these proteins of M. gallisepticum in adhesion to the host cells is discussed.     

Elongation factor (EF-Tu) gene probe detects polymorphism in Mycoplasma strains

Abstract Polymorphism in mycoplasma strains was observed by Southern blot hybridization of the digested mycoplasma DNAs with the elongation factor (EF-Tu) gene tuf of Escherichia coli . The hybridization patterns revealed genotypic heterogeneity among Mycoplasma gallisepticum strains and a remarkable degree of homogeneity among Mucoplasma pneumoniae strains isolated from pneumonia patients. The distinction among M. gallisepticum strain clusters achieved by the tuf gene probe corresponded exactly with that obtained with the rRNA gene probe pMC5. The tuf gene probe may thus be added as another effective tool in the taxonomy of Mollicutes and in epidemiological surveys of mycoplasma infections.     

Strain identification in Rhizobium trifolii using DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistances

Abstract Indigenous strains of R. trifolii , originating from different geographical regions, were compared using total DNA restriction analysis, plasmid DNA profiles and intrinsic antibiotic resistance patterns. The results of strain relatedness using 2 of these techniques (DNA restriction and antibiotic resistance patterns) were similar, and indicated that these strains formed a heterogeneous group. A large variation in plasmid DNA profiles was observed among the R. trifolii isolates investigated, indicating heterogeneity of plasmid DNA pools. This variation occurred even in some strains that were shown to be related on the basis of their antibiotic resistance patterns. The application of these techniques to differentiate indigenous R. trifolii strains is discussed in this report.     

Biochemical properties and whole-cell protein profiles of group G streptococci isolated from dogs

Whole-cell protein profiles obtained by SDS-PAGE were used in conjuction with physiological tests to differentiate strains of Streptococcus canis isolated from dogs. Fermentation of trehalose and lactose, aesculin hydrolysis together with production of β–D–glucuronidase and α–D–galactosidase allowed the demonstration of nine different biotypes. However, visual analysis of the protein patterns andcomparison by the coefficient of Dice showed minor differences in band patterns among strains. Only two different profiles were observed. Although a correlation between biotypingand protein profile has been found, this kind of analysis did not provide the basis for a typing method.     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

Antigenic variants of Mycoplasma gallisepticum major surface lipoprotein, pMGA, are encoded by a large gene family. In this study sequence analyses of the PCR-amplified pMGA genes showed two types of sequences similar to the pMGA1.2 gene in M. gallisepticum strains. They differed in the sequence encoding a proline-rich region (PRR) at the N-terminus of the pMGA protein. The type A genes had sequences similar to the published pMGA1.2 gene sequence of strain S6, whereas the type B genes lacked the second repetitive segment encoding PTPN sequence within PRR and were similar to the published sequence of PG31 strain. Low in vitro passages of M. gallisepticum strains isolated recently in Slovenia from four avian species showed very different expression patterns of pMGA1.2 and pMGA1.9 genes. Among isogenic populations of S6(B) and IHB1 strains a high frequency of pMGA antigenic variants lacking an epitope for monoclonal antibody (mAb) 71 was found. Strain IHB1 clones, which synthesized pMGA recognized by mAb 71, transcribed pMGA genes whose partial sequence encoded the amino acid sequence (262)TNGDEPRSVS of the mAb 71 epitope. Other IHB1 clones synthesized pMGA variants with different isoelectric points, lacking the epitope for mAb 71, but expressing downstream epitopes for other mAbs. Our study suggests that a molecular basis for pMGA antigenic variation lies in the corresponding changes at the DNA level.     

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

ABSTRACT: BACKGROUND: Haemophilus parasuis is the causative agent of Glasser's disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates. RESULTS: The DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson's index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined. CONCLUSIONS: The RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.     

Trypanosoma cruzi: characterization of in vitro and in vivo synthesized polypeptides from epimastigote forms of different strains

Intact RNAs were isolated from epimastigote forms of different Trypanosoma cruzi strains. Translation of the mRNAs using rabbit reticulocyte lysate and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed protein profiles comparable to those observed by labeling cells in vivo. No major interstrain differences were observed in the patterns of the polypeptides synthesized in vitro and in vivo, indicating that metabolic proteins are similar among distinct strains. Several T. cruzi polypeptides produced in the rabbit reticulocyte lysate system were immunoprecipitated by specific antisera. The patterns of antigenic polypeptides recognized by antisera raised against epimastigotes from different strains were also very similar.     

Physiological and Biochemical Characteristics of Iranian Strains of Xanthomonas axonopodis pv. citri, the Causal Agent of Citrus bacterial Canker Disease

Twenty-four strains of Xanthomonas axonopodis pv. citri ( Xac ), the causal agent of bacterial canker of citrus, isolated from Mexican lime ( Citrus aurantifolia ) and lemon ( Citrus limon ) in southern Iran, were characterized phenotypically. Strains were all pathogenic on C. aurantifolia . Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis revealed slight differences in soluble protein profiles among the strains. Based on host range specificity and phenotypic characteristics, representative strains were differentiated into two groups of Asiatic (A) and atypical Asiatic (aA) forms. DNA fingerprinting analysis using Eco RI as the restriction endonuclease showed a negligible difference in restriction pattern between the two groups. On the basis of isozymic analysis, the two groups were distinct with respect to superoxide dismutase (SOD) and esterase (EST) banding patterns. Plasmid DNA profile analysis showed that the bacterial strains were different from each other in terms of plasmid number and molecular weight. Phage typing study revealed that most of group A strains were susceptible to Cp1 and/or Cp2 and some were resistant to both phage types including the strain in aA group. Bacteriocin production test indicated that there was a variation among Xac strains using different indicators for each bacteriocin producer. It is concluded that the Iranian strains of Xac are heterogeneous and constitute a subgroup(s) within the pathotype A.     

Species attribution and distinguishing strains of Oenococcus oeni isolated from Chinese wines

Summary Twenty-four strains of Oenococcus oeni were isolated from different Chinese wines. Differentiation of isolates was carried out by analysis of total soluble cell protein patterns and random amplified polymorphic DNA (RAPD) patterns. The results indicated that the total soluble cell protein patterns could be used to distinguish different genera but fail to distinguish different strains. It was also found that strain RAPD pattern can successfully distinguish isolates by UPGMA analysis. The RAPD profiles (107 different prints) were strain specific and two main groups of strains were screened.     

The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes

Electrophoretic banding pattens of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains ofSaccharomyces cerevisiae used for commercial production of wine. These fingerprinting procedures provided unique profiles for all the different yeast strains and can therefore be used to identify and control industrial strains. Furthermore, the protein profiles, restriction fragments banding patterns and electrophoretic karyotyping by contour clamped homogeneous electric field electrophoresis (CHEF), were valuable to differentiate hybrid and parental strains in yeast breeding programmes. Hybrid strains, with desirable oenological properties, were obtained by mass spore-cell mating between a heterothallic killer yeast and two homothallic sensitive strains and all were shown to have unique DNA fingerprints and electrophoretic karyotypes.     

Comparison of Staphylococcus spp. cellular and extracellular proteins by SDS-PAGE

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of 15 strains representing 10 Staphylococcus species showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS-PAGE method.     

Characterization of cross-reacting serotypes of Campylobacter jejuni

Some strains of Campylobacter jejuni react with more than one reference antiserum from the serotyping scheme based on heat-stable lipopolysaccharide antigens. To investigate the molecular basis of these cross-reactions, lipopolysaccharides from the reference strains for serotypes 4, 13, 16, 43, and 50 and isolates recovered during two different outbreaks of C. jejuni enteritis were analyzed by passive haemagglutination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis coupled with silver staining or immunoblotting. The results showed that lipopolysaccharides from the reference strains and the isolates reacted with antisera prepared against heterologous strains in various combinations and that both silver-stainable, low Mr and non-silver-stainable, high Mr lipopolysaccharide components provided the antigenic determinants associated with the cross-reactions. There were strain-to-strain differences in the structural and antigenic properties of these macromolecules and shared antigenic determinants were not always provided by a common structure. Analysis of the silver-stained lipopolysaccharide profiles, outer membrane protein patterns, and chromosomal DNA restriction patterns indicated that strains with the same lipopolysaccharide profile could have the same outer membrane protein pattern and the same DNA restriction pattern. These results provided evidence for the presence of clones within this antigenic complex and implicated antigenic variation in some strains as the phenomenon responsible for the multiplicity of cross-reactions.     

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.     

Evaluation of random amplified polymorphic DNA (RAPD)-PCR as a method to differentiate Lactobacillus acidophilus,Lactobacillus crispatus,Lactobacillus amylovorus,Lactobacillus gallinarum,Lactobacillus gasseri,and Lactobacillus johnsonii

The technique random amplified polymorphic DNA (RAPD)-PCR was evaluated as a method to differentiate Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus amylovorus, Lactobacillus gallinarum, Lactobacillus gasseri, and Lactobacillus johnsonii. Representative strains, including the type of each species, were selected from different clusters obtained by numerical analysis of total soluble cell protein patterns. Results obtained by RAPD-PCR corresponded well with results obtained by numerical analysis of total soluble cell protein patterns. The type strains of each species displayed different RAPD profiles. Strains with identical L(+)- nicotinamide adenine dinucleotide-dependent lactic dehydrogenase (nLDH) electrophoretic profiles could be distinguished on the basis of their RAPD profiles.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.