Oligonucleotide probes to the 16s ribosomal RNA: implications of sequence homology and secondary structure with particular reference to the oral species Prevotetla intermedia and Prevotetla nigrescens

Abstract

Eight oligonucleotides based upon regions of the small subunit 165 ribosomal RNA gene sequences were analysed against a background of their position within the molecule and their two‐dimensional structure to rationalise their use in recognising Prevotetla intermedia and Prevotetla nigrescens. The 41 clinical isolates from both oral and respiratory sites and two reference strains were subjected to DNA‐DNA hybridisation and multilocus enzyme etectrophoresis to confirm their identity. Alignment of oligonucleotide probes designated I Bi‐2 to I Bi‐6 (for P. intermedia) and 2Bi‐2 (for P. nigrescens) with the 165 rRNA suggested that these probes lacked specificity or were constructed from hypervariable regions. A 52–mer oligonucleotide (designated Bi) retiably detected both species. Because of the high degree of concordance between the I 65 rRNAs of both species, it was necessary to vary the stringency of hybridisation conditions for detection of both species. Thus probe I Bi‐I recognised P. intermedia while I Bi‐l detected both P. intermedia and P. nigrescens at low stringency. However, under conditions of high stringency only P. nigrescens was recognised by probe 2Bi‐I. These probes were highly specific and did not hybridise with DNA from the closety retated P. corporis, nor other periodontal pathogens such as Fuso‐bacterium nucleatum, Actinobacillus actinomycetemcomit‐ans, Treponema denticola and several pigmented species such as Prevotetla metoninogenica, P. denticola, P. loescheii, Porphyramonas osaccharolytica, Py. endodontalis, Py. gin‐givalis, Py. levii, and Py. macacae.

Detection of antibodies against Fusobacterium necrophorum and Porphyromonas levii-like species in dairy cattle with papillomatous digital dermatitis

“…We developed a culture method that improved the rate of isolation of treponemes. During this study, colonies producing black pigment were isolated frequently from the primary cultures, suggesting that Porphyromonas species, obligate anaerobic Gram‐negative bacteria which were previously classified as Bacteroides ( Prevotella ) species (15), were present in the lesions. Moreover, the 16S rRNA genes of Fusobacterium necrophorum and Porphyromonas levii‐ like species were detected in the PDD lesions (16).…”

“…Because F. necrophorum and Bacteroides ( Prevotella ) species (now more commonly known as Porphyromonas species, including P. levii , which was previously classified as a subspecies of Bacteroides ( Prevotella ) melaninogenicus ) (15, 17) have consistently been found to be associated with bovine foot diseases–foot rot (18, 33) and IDD (19, 20), they are considered to be the main casual pathogens of these foot infectious diseases. The 16S rRNA gene sequence of our strain HD35‐3 isolated from a PDD lesion had 99% identity with that of P. levii clone 5117 16S ribosomal RNA gene (GenBank accession no.…”