Neil S. Jensen

Serpulina pilosicoli sp. nov., the agent of porcine intestinal spirochetosis.

Phenotypic and genetic traits of porcine intestinal spirochete strain P43/6/78T (= ATCC 51139T) (T = type strain), which is pathogenic and weakly beta-hemolytic, were determined in order to confirm the taxonomic position of this organism and its relationships to previously described species of intestinal spirochetes. In BHIS broth, P43/6/78T cells had a doubling time of 1 to 2 h and grew to a maximum cell density of 2 x 10(9) cells per ml at 37 to 42 degrees C. They hydrolyzed hippurate, utilized D-glucose, D-fructose, sucrose, D-trehalose, D-galactose, D-mannose, maltose, N-acetyl-D-glucosamine, D-glucosamine, pyruvate, L-fucose, D-cellobiose, and D-ribose as growth substrates, and produced acetate, butyrate, ethanol, H2, and CO2 as metabolic products. They consumed substrate amounts of oxygen and had a G+C content (24.6 mol%) similar to that of Serpulina hyodysenteriae B78T (25.9 mol%). Phenotypic traits that could be used to distinguish strain P43/6/78T from S. hyodysenteriae and Serpulina innocens included its ultrastructural appearance (each strain P43/6/78T cell had 8 or 10 periplasmic flagella, with 4 or 5 flagella inserted at each end, and the cells were thinner and shorter and had more pointed ends than S. hyodysenteriae and S. innocens cells), its faster growth rate in liquid media, its hydrolysis of hippurate, its lack of beta-glucosidase activity, and its metabolism of D-ribose. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used revealed that P43/6/78T was related to, but was genetically distinct from, both S. hyodysenteriae B78T (level of sequence homology, 25 to 32%) and S. innocens B256T (level of sequence homology, 24 to 25%). These and previous results indicate that intestinal spirochete strain P43/6/78T represents a distinct Serpulina species. Therefore, we propose that strain P43/6/78 should be designated as the type strain of a new species, Serpulina pilosicoli.

Reclassification of Treponema hyodysenteriae and Treponema innocens in a New Genus, Serpula gen. nov., as Serpula hyodysenteriae comb. nov. and Serpula innocens comb. nov.

The intestinal anaerobic spirochetes Treponema hyodysenteriae B78T (T = type strain), B204, B169, and A-1, Treponema innocens B256T and 4/71, Treponema succinifaciens 6091T, and Treponema bryantii RUS-1T were compared by performing DNA-DNA reassociation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins, restriction endonuclease analysis of DNA, and 16S rRNA sequence analysis. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used showed that T. hyodysenteriae B78T and B204 had 93% sequence homology with each other and approximately 40% sequence homology with T. innocens B256T and 4/71. Both T. hyodysenteriae B78T and T. innocens B256T exhibited negligible levels of DNA homology (less than or equal to 5%) with T. succinifaciens 6091T. The results of comparisons of protein electrophoretic profiles corroborated the DNA-DNA reassociation results. We found high levels of similarity (greater than or equal to 96%) in electrophoretic profiles among T. hyodysenteriae strains, moderate levels of similarity (43 to 49%) between T. hyodysenteriae and T. innocens, and no detectable similarity between the profiles of either T. hyodysenteriae or T. innocens and those of T. succinifaciens, T. bryantii, and Escherichia coli. Restriction endonuclease analysis of DNA was not useful in assessing genetic relationships since there was heterogeneity even between strains of T. hyodysenteriae. Partial 16S rRNA sequences of the intestinal spirochetes were determined by using a modified Sanger method and were compared in order to evaluate the phylogenetic relationships among these and other spirochetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of Serpulina species by NADH oxidase gene (nox) sequence comparisons and nox-based polymerase chain reaction tests

The NADH oxidase genes (nox) of 18 strains of intestinal spirochaetes were partially sequenced over 1246 bases. Strains examined included 17 representatives from six species of the genus Serpulina, and the type strain 513A(T) of the human intestinal spirochaete Brachyspira aalborgi. Sequences were aligned and used to investigate phylogenetic relationships between the organisms. Nox sequence identities between strains within the genus Serpulina were within the range 86.3-100%, whilst the nox gene of B. aalborgi shared between 78.8-83.0% sequence identity with the nox sequences of the various Serpulina strains. A phenogram produced based on sequence dissimilarities was in good agreement with the current classification of species in the genus Serpulina, although an atypical strongly beta-haemolytic porcine strain (P280/1), previously thought to be S. innocens, appeared distinct from other members of this species. Primer pairs were developed from the nox sequence alignments for use in polymerase chain reaction (PCR) identification of the pathogenic species S. hyodysenteriae (NOX1), S. intermedia (NOX2), and S. pilosicoli (NOX3), and for the combined non-pathogenic species S. innocens and S. murdochii (NOX4). The PCRs were optimised using 80 strains representing all currently described species in the genus Serpulina, as well as the type strain of B. aalborgi. Tests NOX1 and NOX4 specifically amplified DNA from all members of their respective target species, whilst tests NOX2 and NOX3 were less sensitive. NOX2 amplified DNA from all 10 strains of S. intermedia from pigs but from only 4 of 10 strains from chickens, whilst NOX3 amplified DNA from only 18 of 21 S. pilosicoli strains, even at low stringency. Tests NOX1 and NOX4 should prove useful in veterinary diagnostic laboratories, whilst NOX2 and NOX3 require further refinement.

Denitrobacterium detoxificans gen. nov., sp. nov., a ruminal bacterium that respires on nitrocompounds

A new group of anaerobic, Gram-positive, high G + C (56-60 mol%) bacteria was isolated from the bovine rumen. Of four strains characterized, all were non-motile and none produced spores. The isolates did not produce indole or H2S and did not hydrolyse gelatin. Cells of each strain exhibited similar rod-shaped morphology (0.5-1.0 x 1.0-1.5 microns) although bulbous ends were sometimes present. None of the four strains were able to grow via oxidation of a variety of potentially fermentable substrates but rather obtained energy for growth via anaerobic respiration processes, oxidizing hydrogen, formate or lactate for reduction of various oxidized nitrogen compounds. Trimethylamine oxide and DMSO were also used as electron acceptor. All four strains shared greater than 99% 16S rRNA gene sequence identity. The closest match found between the 16S rRNA gene sequence of all four strains, NPOH1T, NPOH2, NPOH3 and MAJ1, to sequences available in GenBank was that of Coriobacterium glomerans (86% sequence similarity), a phenotypically dissimilar anaerobe within the class Actinobacteria. To accommodate these bacteria the creation of a new genus and species, Denitrobacterium detoxificans, for placement within the family Coriobacteriaceae is proposed. The type strain, NPOH1T (ATCC 700546T), grew equally well over a narrow range of incubation temperatures tested (32-39 degrees C).

Identification of the swine pathogen Serpulina hyodysenteriae in rheas (Rhea americana).

Recently intestinal spirochetes were isolated from rheas in Ohio and Iowa with a necrotizing typhlocolitis. These intestinal spirochetes, strains R1 and NIV-1, were characterized and compared with other intestinal spirochetes, including strains of S. hyodysenteriae. Both rhea spirochetes were indole positive, strongly beta-hemolytic, grew under a 1% O2:99% N2 atmosphere, and were morphologically similar to spirochetes in the genus Serpulina. Analysis of rRNA gene restriction patterns (ribotypes), and immunoblots of whole cell proteins, indicated both spirochetes were similar to Serpulina hyodysenteriae strains from swine. Comparisons of nearly complete sequences (> 1458 bases) of the 16S rRNA gene of the two rhea spirochetes with S. hyodysenteriae strains confirmed that rhea spirochetes R1 and NIV-1 were strains of S. hyodysenteriae. These results indicate that S. hyodysenteriae has a broader host range than previously recognized.

Comparison of Serpulina hyodysenteriae B78, the type strain of the species, with other S. hyodysenteriae strains using enteropathogenicity studies and restriction fragment length polymorphism analysis.

The enteropathogenicity of Serpulina hyodysenteriae B78, the type strain of the species, was compared with S. hyodysenteriae B204, a known pathogenic strain, in 7 week-old pigs. Clinical signs of swine dysentery were observed in 11/18 pigs (61.1%) inoculated with S. hyodysenteriae strain B204. However, in pigs inoculated with S. hyodysenteriae B78, only 1/21 (4.8%) of the pigs became infected. The 21 pigs inoculated with strain B78 included four pigs which received 5-fold higher numbers of S. hyodysenteriae B78 cells than were normally used in test inoculations. None of the four pigs became infected. Restriction fragment length polymorphism (RFLP) analysis, using a randomly cloned piece of S. hyodysenteriae B204 genomic DNA as the probe (pSRM5), was found to be useful in distinguishing S. hyodysenteriae strains. RFLP analysis confirmed that the one S. hyodysenteriae B78-inoculated pig that exhibited clinical signs of swine dysentery was infected with S. hyodysenteriae B78, and not by contamination with S. hyodysenteriae B204. These results indicate that S. hyodysenteriae B78 is only weakly pathogenic and should not be used in experimental infections of swine or in studies of virulence determinants where use of a pathogenic strain of S. hyodysenteriae would be crucial.