ABSTRACT
The nucleotide sequences of the DNA gyrase B subunit gene (gyrB) of Fusobacterium necrophorum subsp. necrophorum, F. necrophorum subsp. funduliforme and F. varium were determined and analyzed together with those of F. nucleatum subsp. nucleatum and F. nucleatum subsp. vincentii. On the phylogenetic tree constructed, the strains of each fusobacterial species formed distinct clusters with deep sublines. The degree of sequence similarity within each cluster was 93.2% or more, whereas similarities between clusters ranged from 70.1 to 72.7%. These clusters were recovered with 100% bootstrap probabilities and are in very good agreement with the species of Fusobacterium. These data suggest that gyrB is an accurate genealogical marker for the classification of the fusobacterial taxa considered in this study.
Subject(s)
DNA Gyrase/genetics , Fusobacterium/genetics , Phylogeny , Base Sequence , Cluster Analysis , DNA Primers , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology , Species SpecificityABSTRACT
The phylogenic relationships of two subspecies of Fusobacterium necrophorum were investigated by randomly amplified polymorphism DNA-polymerase chain reaction (RAPD-PCR). With each of the 12 random primers, the DNA fingerprints generated were subjected to cluster analysis for dendrograms. The analysis indicated that twelve strains were organized into two major clusters, and that all strains of each subspecies were confined to one cluster. Furthermore, two of the random primers examined each generated a unique band in F. n. necrophorum strains. We cloned these specific bands and determined the nucleotide sequences. A search for amino acid sequence homologies revealed that the two specific fragments had significant homology to the rpoB gene of Lactococcus lactis subsp. lactis and the hemagglutinin-related protein gene of Ralstonia solanacearum, respectively. New specific primers designed for the rpoB gene were able to amplify 900bp fragments from both subspecies. However, the specific primers designed for the hemagglutinin-related protein gene amplified only a 250bp fragment of the genome of the F. n. necrophorum strains, suggesting that this gene is unique to F. n. necrophorum. These results were further confirmed by dot blot hybridization. Finally, a one-step duplex PCR technique in a single tube for the rapid detection and differentiation of the F. necrophorum subspecies was developed.
Subject(s)
DNA, Bacterial/genetics , Fusobacterium necrophorum/classification , Base Sequence , Cloning, Molecular , Cluster Analysis , DNA Primers/chemistry , DNA Primers/genetics , DNA, Bacterial/chemistry , Fusobacterium necrophorum/chemistry , Fusobacterium necrophorum/genetics , Molecular Sequence Data , Nucleic Acid Hybridization , Phylogeny , Polymerase Chain Reaction/veterinary , Random Amplified Polymorphic DNA Technique/veterinary , Sequence Analysis, DNAABSTRACT
The virulence of 5 strains of M. intracellulare and 6 strains of M. avium to mice were examined. The former bacteria were obtained from the patients with mycobacterial pulmonary disease and the latter were from AIDS patients respectively. C57BL/6 (NRAMP-1 susceptible) and its NRAMP-1 congenic mice (resistant) were used to evaluate the virulence of these bacteria. Three of the 5 strains of M. intracelllulare showed a relatively high virulence. They grew in the liver, spleen and lungs of susceptible mice and even in the lungs of resistant mice. On the other hand, none of 6 strains of M. avium could grow in either susceptible or resistant mice. In conclusion, our mouse model may be a useful tool for evaluating the virulence of bacteria isolated from mycobacteriosis patients.
Subject(s)
Mycobacterium avium Complex/pathogenicity , Mycobacterium avium/pathogenicity , Acquired Immunodeficiency Syndrome/microbiology , Animals , Humans , Liver/microbiology , Lung/microbiology , Mice , Mice, Congenic , Mice, Inbred C57BL , Mycobacterium avium/growth & development , Mycobacterium avium/isolation & purification , Mycobacterium avium Complex/growth & development , Mycobacterium avium Complex/isolation & purification , Mycobacterium avium-intracellulare Infection/microbiology , Spleen/microbiology , VirulenceABSTRACT
Six strains of Trichophyton verrucosum were used in a test to determine effects of urea and sodium nitrate on their growth. Despite having urease activity, the strains failed to utilize the urea and sodium nitrate for their growth. Moreover, the organisms were killed if large amounts of these nitrogen compounds and ammonia were added to the medium. These nitrogen compounds, distributed in cattle breeding soil, inhibited the growth of T. verrucosum. Thus, soil in cattle breeding environments does not seem to be a reservoir of this pathogen.
Subject(s)
Ammonia/pharmacology , Nitrates/pharmacology , Trichophyton/growth & development , Urea/pharmacology , Animals , Cattle , Hydrogen-Ion Concentration , Trichophyton/drug effects , Trichophyton/physiologyABSTRACT
The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.
Subject(s)
DNA, Ribosomal Spacer/genetics , Fusobacterium necrophorum/genetics , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Base Sequence , DNA, Ribosomal Spacer/chemistry , Fusobacterium necrophorum/chemistry , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 23S/chemistry , Sequence Homology, Nucleic AcidABSTRACT
The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level.
Subject(s)
DNA, Ribosomal Spacer/genetics , Fusobacterium/classification , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Base Sequence , Blotting, Southern , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal Spacer/chemistry , Fusobacterium/chemistry , Fusobacterium/genetics , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 23S/chemistry , Sequence AlignmentABSTRACT
Tumor necrosis factor-alpha (TNF-alpha) has been reported to be involved in the development and progression of acquired immunodeficiency syndrome (AIDS). To study the role of this cytokine in AIDS pathogenesis, we constructed a chimeric simian and human immunodeficiency virus (SHIV) having the human TNF-alpha gene (SHIV-TNF) and characterized its properties in vitro. SHIV-TNF replicated both in M8166, a human T cell line, and in monkey peripheral blood mononuclear cells (PBMCs). Along with SHIV-TNF replication, TNF-alpha was detected in the culture supernatant by ELISA. The maximum expression level of TNF-alpha reached 120 ng/ml in M8166 cells, and 2.5 ng/ml in monkey PBMCs. The expressed TNF was biologically active, as shown by a cytotoxic assay using TNF-sensitive L929 mouse fibroblasts. This activity was detected at least until 10 passages of SHIV-TNF (74 days after the initial infection). In monkey PBMCs, SHIV-TNF replicated much better than the parental SHIV-NI. Flow cytometric analysis showed that the death of monkey PBMCs infected with SHIV-TNF was severer than that caused by the parental SHIV-NI. These results suggest that SHIV-TNF would be useful for inducing the disease in a monkey model, which may contribute to a better understanding of the role of TNF-alpha in AIDS etiology.
