Relationships among strains classified with the ruminant Pasteurella haemolytica-complex using quantitative evaluation of phenotypic data

The phenotypic relationships among 246 trehalose-negative strains classified under the [Pasteurella] haemolytica-complex in ruminants were investigated by clustering and multidimensional ordinations based upon 79 phenotypic characters. A quantitative evaluation of phenotypic data using a 5-level scoring system is presented permitting a comprehensive utilization of the recorded phenotypic variation among the strains in the analyses. Clustering and ordination analyses display complementary aspects of data which has been clearly demonstrated in this investigation. The main clusters revealed by the numerical techniques could be related to distinctive phenotypic differences and showed an extensive correlation with the recognized biogroups. This classification was based only upon 4 characters (fermentation of L-arabinose, D-sorbitol, glucosides and ornithine decarboxylase). In contrast, there was no obvious interpretation of the clusters formed by using binary scores. Phenotypic subgroups within the recognized biogroups have been described as well as a new, related group of bacteria, tentatively named Bisgaard taxon 36. Quantitative interpretation of phenotypic data seems to represent a promising method for finding relations among affiliated strains of bacteria and to assist in forming hypotheses for subsequent genotypic investigations.     

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.     

Polyphasic taxonomic study of clinically significant actinomadurae including the description of Actinomadura latina sp.nov

Thirty-one strains received either as Actinomadura madurae or Actinomadura pelletieri were assigned to four phena, clusters 1 to 4, in a numerical phenetic survey of the genus Actinomadura. Clusters 2 and 4 corresponded to the validly described species A. madurae and A. pelletieri whereas clusters 1 and 3 encompassed strains received as A. madurae and A. pelletieri, respectively. The two clusters that contained A. madurae strains formed a single taxon when a dataset lacking the antibiotic sensitivity entries was examined. Results from pyrolysis mass spectrometric and DNA amplification fingerprinting analyses underpinned the taxonomic status of clusters 2, 3 and 4 and suggested that the A. madurae strains accounted for a relatively wide range of variation. It is proposed that the cluster 3 strains be given species status within the genus Actinomadura given the congruence found between the chemical, molecular and numerical phenetic data. The name Actinomadura latina is proposed for the new taxon. The type strain is DSM 43382.     

Suspected Brazilian purpuric fever in a toddler with overwhelming Epstein-Barr virus infection

We describe a toddler from Connecticut who developed purulent conjunctivitis, fever, and a morbilliform rash. Blood cultures were positive for Haemophilus influenzae biogroup aegyptius; further investigation was performed to assess the possibility that the illness was consistent with Brazilian purpuric fever, which, to our knowledge, has not been reported in the United States. This isolate shared morphological and some biochemical characteristics with previously studied H. influenzae biogroup aegyptius strains but differed according to slide agglutination testing, plasmid characterization, and ribotyping. Blood and tissue samples obtained during his hospitalization were also positive for Epstein-Barr virus. The child died 8 days after hospitalization. Fifty other cases of invasive H. influenzae infection were identified by active surveillance studies. Of the 49 viable surveillance isolates, 10 were biotype III (two of which had the same ribotype as the strain from our case.     

The effect of LH, FSH and pregnant mares' serum gonadotrophin on ornithine decarboxylase activity in thecal and granulosa tissue during follicular growth and atresia in laying hens (Gallus domesticus)

The effect of ovine LH, porcine FSH and pregnant mares' serum gonadotrophin (PMSG) on the activity of ornithine decarboxylase activity in theca and granulosa tissue during folliculogenesis in laying hens is described. The changes in the activity of ornithine decarboxylase induced by hormonal challenge was used to measure the sensitivity of the tissue to the hormone. Thecal tissue from small (< 6 mm) follicles showed a large increase in the activity of ornithine decarboxylase 3 h after treatment with LH, FSH and PMSG, in vivo, whereas ornithine decarboxylase activity in thecal tissue from large (> 8 mm) preovulatory follicles and atretic follicles did not respond to any of the hormonal treatments. Ornithine decarboxylase activity in granulosa tissue from the largest preovulatory follicle increased significantly 3 h after treatment with LH and PMSG in vivo; no effect was observed with FSH. Granulosa tissue from the third largest and fifth largest preovulatory follicles were refractory to the hormonal treatments. Basal activity of ornithine decarboxylase in granulosa tissue from preovulatory follicles increased as the follicles approached ovulation, whereas the activity in thecal tissue from the same follicles decreased. The difference in sensitivity of thecal tissue from small and large preovulatory follicles towards gonadotrophin treatment in vivo is correlated with the difference in the observed rate of atresia occurring within the two groups of follicles. Atresia is the common fate for small follicles, whereas it is a rare event for large preovulatory follicles under normal physiological conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Generation of depth-perception information in stereoscopic nuclear magnetic resonance imaging by non-linear magnetic field gradients

This study was undertaken to characterize serial Haemophilus parainfluenzae strains from epidemiologically unrelated chronic obstructive pulmonary disease (COPD) patients and from healthy carriers. A comprehensive approach was used including different phenotypical and molecular typing methods: biotyping, antibiotyping, conventional ribotyping, pulsed field gel electrophoresis (PFGE) assay, and PCR-ribotyping. Conventional ribotyping and PFGE analysis were confirmed as excellent procedures to differentiate isolates of the same species and biotype. Conversely, in our study, PCR-ribotyping proved to be suitable for taxonomic purposes, unambiguously identifying H. parainfluenzae from H. influenzae but not discriminating strains at the intraspecific level for epidemiological typing. Phylogenetic analysis of restriction fragment length polymorphism (RFLP) data of sequences related to the rrn operon demonstrated that H. parainfluenzae strains associated to COPD are spread among many diverging lineages.     

Unique organization of the CTX genetic element in Vibrio cholerae O139 strains which reemerged in Calcutta, India, in September 1996

We studied the restriction fragment length polymorphism of the rRNA gene and CTX genetic element in Vibrio cholerae O139 Bengal, which resurged in Calcutta in September 1996 after a gap of 32 months. While the strains from this resurgence were indistinguishable from the earlier strains by ribotyping, the structure of the CTX genetic element present in the current O139 strains was found to be unconventional.     

Immunochemical demonstration of increased accumulation of ornithine carboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.     

Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity

Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).     

Ornithine decarboxylase in human brain: influence of aging, regional distribution, and Alzheimer's disease

Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by approximately 60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (-70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.     

Identification and typing of hospital strains of Acinetobacter calcoaceticus-Acinetobacter baumanni complex

A collection of 95 strains of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex, isolated between 1991 and 1993 in the Prague Burn Center (BC), was studied. Ninety-one strains were isolated from 43 patients: 50 of them from burnt sites, 22 from endotracheal tube, 13 from urine, 3 from blood and 3 from venous catheter, and 4 strains were isolated from the hospital environment and the nursing staff. The strains were classified by restriction endonuclease fingerprinting of total DNA, plasmid profile analysis, ribotyping, comparison of antibiograms, biotyping and according to epidemiological data, into 31 relatedness groups each of them including 1 to 29 strains, likely to be isolates of the same strain. None of the methods used enabled to distinguish all groups. The importance of the polyphasic approach is emphasized since three multiresistant strains, isolated almost simultaneously in the BC, needed at least two methods to be distinguished (e.g. ribotyping and biotyping). Twenty-eight representative strains of different groups were identified by ribotyping: 18 of them were allocated to genomospecies 2 (A. baumannii), 5 to genomospecies 3 and 5 to genomospecies 13 sensu Tjernberg and Ursing. Only A. baumannii was found to spread among patients. Strains of two multiresistant groups persisted in the BC throughout the period studied and strains of one of these groups were responsible for an outbreak in the autumn of 1993. The methods mentioned above were used to describe 12 multiresistant strains isolated in three hospital wards in other localities. When ribotyped these strains were identified as A. baumannii. The strains of the same origin were identical in their typing profiles while the strains of different origins were easy to differentiate using any of the above methods; nevertheless, 2 of these groups were almost identical to 2 groups of multiresistant strains isolated in the BC.     

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.     

Genetic heterogeneity of clinical strains of Yersinia enterocolitica traced by ribotyping and relationships between ribotypes, serotypes, and biotypes

A series of 74 Yersinia enterocolitica clinical strains collected in a Spanish region and 10 reference strains, assigned to nine serotypes and five biotypes, were analyzed by ribotyping procedures. Riboprobing, performed separately with HindIII and BglI and using an rrn operon as the probe, generated 13 and 11 ribotypes (discrimination index [DI] = 0.56 and 0.55), respectively. PCR ribotyping, performed with primers complementary to conserved regions of 16S and 23S rRNA genes, generated 13 ribotypes (DI = 0. 56). A combination of data from the three procedures allowed for further discrimination into 17 combined ribotypes (DI = 0.83). The dendrogram obtained by cluster analysis of data from riboprobing indicated a high heterogeneity of the ribosomal DNA regions of the strains under study (similarities between 10 and 92%), which were grouped into three clusters at a similarity level of 0.32. The major cluster included 10 branches, and 7 of these formed a subcluster (similarity coefficient, >83%) represented by strains of serotype O:3 and biotype 2, 3, or 4. The second cluster included four branches, represented by strains belonging to seven non-O:3 serotypes, biotypes 1A and 2, and two of these branches included pyrazinamidase-positive as well as pyrazinamidase-negative strains. The remaining three branches, represented by O:3-biotype 4 strains, formed a third cluster weakly related to the others. Data from this study showed that Y. enterocolitica O:3 organisms assigned to a prevalent and endemic lineage and non-O:3 organisms assigned to three other less-frequent lineages are circulating and causing human disease in the Spanish region under study.     

Characterization of a second lysine decarboxylase isolated from Escherichia coli

We report here on the existence of a new gene for lysine decarboxylase in Escherichia coli K-12. The hybridization experiments with a cadA probe at low stringency showed that the homologous region of cadA was located in lambda Kohara phage clone 6F5 at 4.7 min on the E. coli chromosome. We cloned the 5.0-kb HindIII fragment of this phage clone and sequenced the homologous region of cadA. This region contained a 2,139-nucleotide open reading frame encoding a 713-amino-acid protein with a calculated molecular weight of 80,589. Overexpression of the protein and determination of its N-terminal amino acid sequence defined the translational start site of this gene. The deduced amino acid sequence showed 69.4% identity to that of lysine decarboxylase encoded by cadA at 93.7 min on the E. coli chromosome. In addition, the level of lysine decarboxylase activity increased in strains carrying multiple copies of the gene. Therefore, the gene encoding this lysine decarboxylase was designated Idc. Analysis of the lysine decarboxylase activity of strains containing cadA, ldc, or cadA ldc mutations indicated that ldc was weakly expressed under various conditions but is a functional gene in E. coli.     

Development of rRNA-targeted PCR and in situ hybridization with fluorescently labelled oligonucleotides for detection of Yersinia species

In this report, we present details of two rapid molecular detection techniques based on 16S and 23S rRNA sequence data to identify and differentiate Yersinia species from clinical and environmental sources. Near-full-length 16S rRNA gene (rDNA) sequences for three different Yersinia species and partial 23S rDNA sequences for three Y. pestis and three Y. pseudotuberculosis strains were determined. While 16S rDNA sequences of Y. pestis and Y. pseudotuberculosis were found to be identical, one base difference was identified within a highly variable region of 23S rDNA. The rDNA sequences were used to develop primers and fluorescently tagged oligonucleotide probes suitable for differential detection of Yersinia species by PCR and in situ hybridization, respectively. As few as 10(2) Yersinia cells per ml could be detected by PCR with a seminested approach. Amplification with a subgenus-specific primer pair followed by a second PCR allowed differentiation of Y. enterocolitica biogroup 1B from biogroups 2 to 5 or from other pathogenic Yersinia species. Moreover, a set of oligonucleotide probes suitable for rapid (3-h) in situ detection and differentiation of the three pathogenic Yersinia species (in particular Y. pestis and Y. pseudotuberculosis) was developed. The applicability of this technique was demonstrated by detection of Y. pestis and Y. pseudotuberculosis in spiked throat and stool samples, respectively. These probes were also capable of identifying Y. enterocolitica within cryosections of experimentally infected mouse tissue by the use of confocal laser scanning microscopy.     

Taxonomic aspects of strains from Patagonia and type strains of Desulfovibrio

Fourteen type strains and 42 indigenous strains of Desulfovibrio were studied and taxonomic numeric methods were applied (Cluster Analysis, Principal Coordinates Analysis, Correspondence Analysis). The type strains as well as the indigenous ones share a low quantity of carbon and energy sources. Type strains have a taxonomic structure which shows net clusters; indigenous strains possess a grading (or "adjustment") in their positive reactions hence the taxonomic structure is not so clear. Both classifications are firm since they suffer minimal changes when they are joined.     

Relative in vitro stability of ornithine decarboxylase from liver preneoplastic nodules and hepatomas

A very rapid and drastic microsome-dependent in vitro inactivation of the hydrocortisone-induced ornithine decarboxylase in rat liver was reported recently (M. F. Zuretti and E. Gravela, Biochim. Biophys. Acta. 742: 269-277, 1983). Present results show that ornithine decarboxylase from preneoplastic nodules and hepatomas, which have been induced in rats by N-2-fluorenylacetamide, is much more stable, its greater stability not being accounted for by a lower microsome-bound inactivating capacity. The possibility of a relationship between the in vitro enzyme stability and the increase of enzyme activity in neoplastic tissues is suggested.     

Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A. pleuropneumoniae. Amplification and sequence analysis of the 16S-23S rDNA ribosomal intergenic sequence (RIS) from the three species showed the existence of two RIS's, differing by about 100 bp. Both sequences contained a region resembling the ribonuclease III cleavage site found in Escherichia coli. The smaller RIS contained a Glu-tRNA gene, and the larger one contained genes encoding Ile-tRNA and Ala-tRNA. These tRNA's showed a high sequence homology to the respective tRNA genes found in E. coli. Sequence analysis of the RIS's showed a high degree of genetic similarity of 24 strains of A. pleuropneumoniae. The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ hybridization experiments to detect A. pleuropneumoniae in biopsies of diseased porcine lungs.