Reappraisal of the taxonomy of Streptococcus suis serotypes 20, 22 and 26: Streptococcus parasuis sp. nov.

In order to clarify the taxonomic position of serotypes 20, 22 and 26 of Streptococcus suis, biochemical and molecular genetic studies were performed on isolates (SUT-7, SUT-286(T), SUT-319, SUT-328 and SUT-380) reacted with specific antisera of serotypes 20, 22 or 26 from the saliva of healthy pigs as well as reference strains of serotypes 20, 22 and 26. Comparative recN gene sequencing showed high genetic relatedness among our isolates, but marked differences from the type strain S. suis NCTC 10234(T), i.e. 74.8-75.7 % sequence similarity. The genomic relatedness between the isolates and other strains of species of the genus Streptococcus, including S. suis, was calculated using the average nucleotide identity values of whole genome sequences, which indicated that serotypes 20, 22 and 26 should be removed taxonomically from S. suis and treated as a novel genomic species. Comparative sequence analysis revealed 99.0-100 % sequence similarities for the 16S rRNA genes between the reference strains of serotypes 20, 22 and 26, and our isolates. Isolate STU-286(T) had relatively high 16S rRNA gene sequence similarity with S. suis NCTC 10234(T) (98.8 %). SUT-286(T) could be distinguished from S. suis and other closely related species of the genus Streptococcus using biochemical tests. Due to its phylogenetic and phenotypic similarities to S. suis we propose naming the novel species Streptococcus parasuis sp. nov., with SUT-286(T) ( = JCM 30273(T) = DSM 29126(T)) as the type strain.

Disruption of srtA gene in Streptococcus suis results in decreased interactions with endothelial cells and extracellular matrix proteins.

Streptococcus suis, a major pathogen of swine, is an emerging zoonotic agent which causes meningitis and septic shock. In this study, we investigated the ability of S. suis mutant strain (SRTDeltaA) lacking the sortase A gene (srtA) to interact with host cells and extracellular matrix (ECM) proteins, as well as its virulence in a mouse infection model. We demonstrated that mutant SRTDeltaA had reduced capacity to adhere to and invade porcine brain microvascular endothelial cells compared to the wild-type strain. In addition, mutant SRTDeltaA also showed significantly less adherence to plasma fibronectin, cellular fibronectin and collagen type I. However, disruption of srtA had little effect on the virulence of S. suis in a mouse intraperitoneal model of infection. These results indicate that surface proteins anchored by sortase A are required for a normal level of bacterial binding. However, other factors may also be important for S. suis virulence and interaction with host tissues.

Sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens.

The nucleotide sequence of the Rhodococcus equi gene encoding the virulence-associated 15-17-kDa antigens, located on plasmid pREAT701, has been determined. The gene encodes a 19-kDa protein of 189 amino acids, with an Ala-rich leader signal sequence (SS). At least five SS peptidase cleavage sites were found in this region. The molecular diversity of 15-17-kDa antigens might be attributed to the multiple SS peptidase cleavage sites.

Demonstration of the third antigenically distinct outer membrane lipoprotein (OmlA) in Actinobacillus pleuropneumoniae serotype 7.

The gene encoding an outer membrane lipoprotein (OmlA) of Actinobacillus pleuropneumoniae strain WF83 (serotype 7 reference strain), designated omlA7, was sequenced. The amino acid sequence of OmlA7 showed 64.5 and 71.6% identity to that of OmlA from serotypes 1 (OmlA1) and 5 (OmlA5), respectively. The first 134 amino acids of OmlA7 were identical to those of OmlA5. A Southern blot analysis revealed the presence of a gene highly homologous to the omlA7 in the reference strains of serotypes 3, 4, 6, and 7. A Western blot analysis using a specific antiserum against a recombinant OmlA7 detected expression of the homologous proteins in the serotypes 4, 6, and 7 reference strains and a serotype 3 field strain, but not in a serotype 3 reference strain. The data demonstrate the third antigenically distinct OmlA is expressed in A. pleuropneumoniae.

Cloning, nucleotide sequence and expression of the gene encoding the cellulose-binding protein 1 (CBP1) of Fibrobacter succinogenes S85.

The nucleotide sequence of the gene encoding the Fibrobacter succinogenes S85 cellulose-binding protein 1 (CBP1) has been determined. The gene encodes a protein of 1054 amino acids with a molecular mass of 118614. The deduced amino acid sequence of CBP1 showed an extensive similarity to the cellulose-binding domain of an endoglucanase (EGCCD) from Clostridium cellulolyticum and contained the reiterated regions. The cloned gene was inserted into an expression vector, pRSETA, and was expressed in E. coli as a fused protein with the peptide consisting of six consecutive histidine residues. The fused protein was detected by immunoblotting using antiserum against CBP1, and exhibited the cellulose-binding activities.

A small cryptic plasmid from Ruminobacter amylophilus NIAH-3 possesses functional mobilization properties.

The complete nucleotide sequence of a small cryptic plasmid designated pRAO1, from the Gram-negative ruminal bacterium Ruminobacter amylophilus NIAH-3, was determined. The plasmid is a circular DNA molecule, 2140 bp in size, with a GC content of 40%. Computer-assisted analysis identified three open reading frames (ORFs), one of which, ORF3 (347 amino acids), displayed a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmid recombination of plasmids from Gram-positive bacteria. We proved the mobilization properties of pRAO1 in the Escherichia coli system using the coresident IncW broad-host-range conjugative plasmid R388. These data demonstrated, for the first time, the mobilization properties of small cryptic plasmids from Gram-negative inhabitants of the rumen.

A specific oligonucleotide probe based on 5S rRNA sequences for identification of Vibrio anguillarum and Vibrio ordalii.

An oligonucleotide DNA probe based on 5S rRNA sequence data was constructed for the identification of the fish pathogens, Vibrio anguillarum and Vibrio ordalii. Specificity of the probe was tested in a colony blot hybridization assay. The respective probe was found to be specific for both V. anguillarum and V. ordalii. No cross hybridization was observed against other fish pathogens and the closely related Vibrionaceae genera. This specific probe may be useful for rapid identification of V. anguillarum and V. ordalii.

Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei.

Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein. Using an Escherichia coli expression library of the C. chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein. DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da. Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains. The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114. However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC. Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice. These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.

Rapid detection and identification of Clostridium chauvoei by PCR based on flagellin gene sequence.

We developed a one-step polymerase chain reaction (PCR) system that specifically detects Clostridium chauvoei. Oligonucleotide primers were designed to amplify a 516-bp fragment of the structural flagellin gene. The specificity of the PCR was investigated by analyzing 59 strains of clostridia, and seven strain of other genera. A 516-bp fragment could be amplified from all the C. chauvoei strains tested, and no amplification was observed by using DNAs from the other strains tested, including Clostridium septicum. Similarly, this PCR-based method specifically detected C. chauvoei DNA sequences in samples of muscle and exudate of obtained from mice within 12h of inoculation. In tests using samples of muscle or liver, the limit of detection was about 200 organisms per reaction. These results suggest that the one-step PCR system may be useful for direct detection and identification of C. chauvoei in clinical specimens.

Effect of growth temperature on maintenance of virulent Rhodococcus equi.

Repeated passage of virulent Rhodococcus equi ATCC 33701 and L1 at 38 degrees C resulted in attenuation of the strains as a result of curing the virulence plasmid; at 30 degrees C, repeated passage had no such effect. At a temperature of 38 degrees C the plasmid-bearing cells replicated more slowly than their plasmid-cured derivatives and so were gradually replaced by cells lacking plasmids. In contrast, at a temperature of 30 degrees C the growth rate of either strain was not affected by the presence or absence of the plasmid. No plasmid-cured derivative was recovered from mouse organs at 48 h after inoculation of a mixture of equal numbers of bacteria with and without plasmids. It is concluded that under nonselective conditions growth temperature is an important factor in maintaining the virulence of R. equi.

Acid-induced autoagglutination found in chicken pathogenic Escherichia coli strain.

Chicken pathogenic Escherichia coli strains were found to autoagglutinate in a static culture of trypticase soy broth (TSB). One strain, designated PDI-386, was further studied for its autoagglutinating property. Acidity in the cultured medium caused by glucose degradation induced the autoagglutination. The bacterial cells grown in a glucose-free L-broth could be aggregated by adding acid, which suggests a potentiality of autoagglutination of the strain grown in the L-broth. The autoagglutinating parent (Agg) formed small colonies with irregular edges like rough colonies on the TS agar, whereas its non-autoagglutinating variant (Nag) formed larger smooth colonies with a perfectly round edge. The Nag colony was easily generated from the Agg colony on the TS agar. The autoagglutinating property was very unstable when the bacteria was passed in the TSB, but rather stable in the L-broth. Under electron microscope, the Agg were found to possess pili of more than 20 microns in length. However, the phenotypic expression of autoagglutination did not correlate with that of mannose-sensitive hemagglutination against guinea pig erythrocytes. Incubation of the Nag in the L-broth at room temperature for more than 10 days provoked the reversion of the autoagglutination. There was no difference between the Agg and the Nag in terms of surface hydrophobicity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of membrane proteins and LPS, and plasmid profiles. The virulence of the Agg was higher than that of the Nag. The autoagglutination property is, however, so unstable that the pathogenicity of E. coli isolates from chickens should be carefully evaluated.

A cryptic DNA sequence, isolated from Actinobacillus pleuropneumoniae, confers a hemolytic activity upon Escherichia coli K12 strains.

A hemolytic recombinant clone on Escherichia coli was obtained from a genomic library of Actinobacillus pleuropneumoniae. The clone possessed a recombinant plasmid, pHLY1, carrying an insert DNA of 1 kilobases. A 21 kilodaltons protein, which is assumed to be a fusion protein with a tetracycline resistant protein of pBR322, was encoded by pHLY1. The nucleotide sequence and its deduced amino acid sequence were different from those of the previously reported hemolysin genes of A. pleuropneumoniae. Furthermore, the amino acid sequence did not show a significant homology with other published sequences in the SWISS-PROT Protein Sequence Data Bank.

DNA sequence of type 1 fimbrin, Fpul1, gene from a chicken pathogenic Escherichia coli serotype O78.

The gene encoding type 1 fimbriae of chicken pathogenic Escherichia coli serotype O78 (designated Fpul1) was cloned and the genetic region encoding fimbrial subunit was sequenced. The nucleotide sequence and its deduced amino acid sequence demonstrated that the Fpul1 was a novel variation among E. coli type 1 fimbriae and showed an extensive homology to previously reported Klebsiella pneumoniae type 1 fimbriae. The E. coli K-12 strains carrying the Fpul1 genes did not show the acid-induced autoagglutination, suggesting that the Fpul1 was genetically distinct from the acid-induced autoagglutination.

Isolation and characterization of type 1 fimbriae from a chicken pathogenic Escherichia coli serotype O78.

Type 1 fimbriae from chicken pathogenic Escherichia coli strain PDI-386 (serotype O78) was purified and characterized. Because of the acid-induced autoagglutination (T. Sekizaki, Y. Nakasato, and I. Nonomura, J. Vet. Med. Sci. 54, 493-499, 1992), the fimbriae could be easily purified by repeating acid sedimentation, washing, and dissolving in buffer (pH 8.0). In electron microscopy, the purified fimbriae showed a filament of 8 nm in diameter and 10 microns in average length. The molecular mass of the protein subunit of the purified fimbriae estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 19,000 daltons. The amino acid composition and its NH2-terminal sequence were similar to the previously described one of the Klebsiella pneumoniae type 1 fimbriae. Moreover, there was an immunological relatedness between them. These results indicated that a molecular diversity found between the fimbriae of E. coli and that of K. pneumoniae has already been existed among chicken pathogenic E. coli strains.

Genetic diversity of the genes encoding the outer membrane lipoprotein (omlA) of Actinobacillus pleuropneumoniae.

An allelic variation of the genes encoding the protective outer membrane lipoprotein (omlA) in Actinobacillus pleuropneumoniae was investigated with polymerase chain reaction (PCR)-restriction fragment length polymorphisms. Primers for PCR were selected from a conserved sequence compared between the omlA genes of A. pleuropneumoniae serotypes 1 and 5a. A DNA fragment of 970 bp was amplified from the genomic DNA of all 12 serotypes of A. pleuropneumoniae. The amplified DNA sequence specifically hybridized under a low stringent condition to the cloned omlA gene of A. pleuropneumoniae. Digestion of the amplified DNA with the enzymes either HinfI or VspI yielded specific polymorphic patterns, allowing discrimination of all serotypes into five distinct groups.

Improved electroporation of Rhodococcus equi.

The condition of an electroporation method was re-evaluated for the introduction of foreign plasmid DNA into Rhodococcus equi. The method is based on an electroporation of the bacteria made competent by culturing in a broth containing glycine and by heat shock at 50 degrees C. Transformation of R. equi could be achieved with a chloramphenicol-resistant shuttle vector originating from Rhodococcus fascians at an efficiency of about 10(4) transformants/microgram DNA. The bacteria were also shown to become competent when they were incubated with a chemical transformation buffer prior to washing with an electroporation buffer.

Development of a PCR test for rapid diagnosis of contagious equine metritis.

In order to establish a rapid diagnostic method for contagious equine metritis (CEM), we developed and evaluated a polymerase chain reaction (PCR) test. Species-specific PCR primer sets were derived from the DNA sequence of a cloned DNA fragment of Taylorella equigenitalis that did not hybridize with the genome of a taxomonically related species, Oligella urethralis. Single step PCR with primer set P1-N2 and two-step semi-nested PCR with primer sets P1-N2 and P2-N2 detected as low as 100 and 10 CFU of the bacteria, respectively. Single-step PCR detected T. equigenitalis from genital swabs of experimentally infected mares with sensitivity comparable to that of bacterial isolation. Furthermore, two-step PCR was more sensitive than the culture method. Upon examination of field samples, 12 out of 3,123 samples were positive by single-step PCR while only 2 were positive by bacterial culture. The 12 PCR-positive samples originated from 5 mares, of which 3 animals were considered to be carriers based on previous bacteriologic and serologic diagnoses for CEM. The PCR test described in this study would provide a specific and highly sensitive tool for the rapid diagnosis of CEM.

Cloning and comparison of heat-stable enterotoxin genes from Escherichia coli strains of bovine, porcine, and avian origins.

The Ent plasmid encoding Escherichia coli heat-stable enterotoxin (ST) isolated from bovine, porcine, and avian strains were used for the cloning of ST genes. The ST+ DNA regions were finally cloned into pBR322 as the 1.75 kilobases PstI fragment. The electron microscopic analysis of self-annealed molecules indicated that ST+ recombinant plasmid had a stem-loop structure of a size the same as that observed in their wild type Ent plasmids. The stem-loop structures and the restriction enzyme cleavage mappings indicated that 4 kinds of ST genes cloned in this experiment may be identical to Tn1681. The ST production levels of the recombinant plasmids were higher than those of the original plasmids.

Nucleotide sequences of the genes for Escherichia coli heat-stable enterotoxin I of bovine, avian, and porcine origins.

The nucleotide sequences of cloned Escherichia coli heat-stable toxin 1 (STI) genes isolated from bovine, avian, and porcine origins were determined. They were found to be almost identical to that of Tn1681. The nucleotide sequences were completely preserved in bovine and avian genes, whereas the porcine gene had different sequences at 3 positions in the external region of STI structural genes, as compared with Tn1681. The amino acid sequences of the STI genes of the 3 animal origins corresponded to STIa, which had initially been found in a bovine strain.

Sequence analysis of a small cryptic plasmid isolated from Streptococcus suis serotype 2.

A small cryptic plasmid designated pSSU1 was isolated from Streptococcus suis serotype 2 strain DAT1. The complete sequence of pSSU1 was 4975 bp and contained six major open reading frames (ORFs). ORF1 and ORF2 encode for proteins highly homologous to CopG and RepB of the pMV158 family, respectively. ORF5 encodes for a protein highly homologous to Mob of pMV158. ORF4 encodes for a protein highly homologous to orf3 of pVA380-1 of S. ferus, but its function is unknown. There was no similarity between ORF3 and ORF6 and other protein sequences. In this plasmid, the ORF1 (CopG protein) was preceded by two multiples of direct repeat and the conserved nucleotides that could be the double-strand origin (DSO) of rolling circle replication (RCR) mechanism. The ORF5 (Mob protein) was followed by a potential hairpin loop that could be the single-strand origin (SSO) of RCR mechanism. The sequence, which was complementary to the leader region of Rep mRNA, was homologous to the countertranscribed RNA (ctRNA) of pLS1. Moreover, a 5-amino acid conserved sequence was found in C terminal of Rep and putative Rep proteins of several pMV158 family plasmids. These observations suggest that this plasmid replicates by use of the rolling circle mechanism.