Serpulina Species Research Articles

Prevalence and disease association of intestinal spirochaetes in chickens in eastern Australia

Faecal samples (n = 1786) from chickens in broiler breeder (n = 28), layer (n = 22) or broiler (n = 19) flocks in the eastern states of Australia were cultured for intestinal spirochaetes. Overall, birds in 42.9% of broiler breeder and 68.2% of layer flocks were colonized with spirochaetes, but no birds in broiler flocks were infected. Colonization rates in infected flocks ranged from 10 to 100% of birds sampled. Faeces from colonized flocks were on average 14% wetter than those from non-colonized flocks. There was a highly significant association between colonization with spirochaetes and the occurrence of wet litter and/or reduced production. A subset of 57 spirochaete isolates from birds in 16 flocks were identified to the species level using a panel of polymerase chain reaction tests. Isolates from nine (56%) of these flocks were spirochaetes that are known to be pathogens of poultry: Serpulina pilosicoli was isolated from birds from five flocks, birds from two flocks were infected with Serpulina intermedia, and in two other flocks both species were identified. Isolates from the other seven flocks belonged to other Serpulina species, which are currently of unknown pathogenicity. This study indicates that infections with intestinal spirochaetes are a common but currently under-diagnosed cause of wet litter and/or reduced egg production in broiler breeder and layer flocks in Australia.

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.

Serpulina alvinipulli sp. nov., a new Serpulina species that is enteropathogenic for chickens.

Strain C1T is an anaerobic spirochaete that causes intestinal disease in chickens. Multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons have indicated that this spirochaete is a Serpulina strain. In these investigations, various phenotypic and genomic properties useful for establishing a taxonomic identity for strain C1T were studied. As determined by electron microscopy, cells of the spirochaete measured 8-11 x 0.22-0.34 mum and had a typical spirochaete ultrastructure. Each cell had 22-30 flagella. C1T cells formed weakly beta-haemolytic colonies on trypticase soy agar plates containing 5% bovine blood. The spirochaete reached maximum population densities of 10(9) cells ml-1 with a 2-4 h population doubling time in brain heart infusion broth containing 10% calf serum (BHIS broth). C1T cultures in BHIS broth were positive in tests for hippurate hydrolysis and negative for indole production. Glucosamine, N-acetyglucosamine, glucose, fructose, maltose and mannose were growth substrates for the spirochaete in heart infusion broth containing 7% calf serum (HS broth). During growth in HS broth beneath an O2/N2 (1:99) atmosphere, cells of the spirochaete consumed O2 and glucose and produced H2, CO2, acetate, butyrate and ethanol. Strain C1T DNA had a G+C content of 24.6 mol%. Based on DNA-DNA hybridization analyses, the DNA of strain C1T exhibited 24-39% relative reassociation with DNA of Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Serpulina murdochii and Serpulina intermedia. These results indicate that chicken spirochaete strain C1T has many phenotypic properties common to Serpulina species and, based on DNA hybridization analysis, represents a unique Serpulina species. For this new species the name Serpulina alvinipulli is proposed, for which the type strain is C1T (= ATCC 51933T).

Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli

Investigations into the possible causes of colitis and typhlocolitis were carried out on 85 pig units in the United Kingdom between 1992 and 1996. Serpulina pilosicoli was identified most commonly,...

Restriction fragment length polymorphism of the periplasmic flagellar flaA1 gene of Serpulina species.

Forty-one reference and field isolates of intestinal spirochetes representing Serpulina hyodysenteriae, Serpulina innocens, Serpulina pilosicoli, Brachyspira aalborgi, and nonclassified weakly beta-hemolytic intestinal spirochetes were compared by restriction fragment length polymorphism (RFLP) of the periplasmic flagellar (PF) flaA1 gene. Six genetically distinct groups (I through VI), each with a unique RFLP fingerprint pattern, were identified by Southern blotting analysis of EcoRV chromosomal DNA digests with a PCR-amplified digoxigenin-labeled 1-kb fragment of the S. hyodysenteriae isolate B78 PF flaA1 gene. The RFLP fingerprint patterns corresponded to known DNA homology differences between Serpulina species and to provisionally designated species described previously by using phenotypic and genotypic classification schemes. RFLP fingerprinting of the PF flaA1 gene provides a relatively simple genotypic method for identification of intestinal spirochetes without the use of radioisotopes.

Recognition of two new species of intestinal spirochetes: Serpulina intermedia sp. nov. and Serpulina murdochii sp. nov.

On the basis of DNA-DNA hybridization data, nine intestinal spirochete strains were grouped into five genospecies. Three of these genospecies were previously recognized Serpulina species, Serpulina hyodysenteriae (type strain, B78), Serpulina innocens (type strain, B256), and Serpulina pilosicoli (type strain, P43/6/78; previously "Anguillina coli"). The other two genospecies were found to be new Serpulina species, for which we propose the names Serpulina intermedia sp. nov. (with type strain PWS/A) and Serpulina murdochii sp. nov. (with type strain 56-150). S. intermedia and S. murdochii cells had a typical spirochete ultrastructure with 22 to 28 periplasmic flagella per cell. Various soluble sugars were growth substrates for S. intermedia and S. murdochii. During growth in basal heart infusion broth supplemented with fetal calf serum beneath an O2-N2 (1:99) atmosphere, cells of these new species consumed oxygen and glucose and produced H2, CO2, acetate, butyrate, and ethanol. The G + C content of the DNA of S. murdochii 56-150T was 27 mol%, and the G + C content of the DNA of S. intermedia PWS/AT was 25 mol%. In addition, a restriction fragment length polymorphism-PCR assay for the detection of intestinal spirochetes was developed. The assay was based on generation and restriction endonuclease analysis (with HinfI, TaqI, Sau3A, and MboII) of a 558-bp amplicon of ribosomal DNA (rDNA) encoding 16S rRNA. The PCR amplification was specific for Serpulina species and Brachyspira aalborgi. Four restriction digest patterns were found for the five Serpulina species. HinfI restriction differentiated S. murdochii and S. innocens from the other species. Sau3A and TaqI restrictions gave unique fragment patterns for S. murdochii and S. pilosicoli, respectively. S. hyodysenteriae and S. intermedia DNAs gave the same fragment pattern regardless of the enzyme tested. B. aalborgi was differentiated from the Serpulina species by MboII digestion of the 16S rDNA amplicon.

Immunoblot reactivity of polyclonal and monoclonal antibodies with periplasmic flagellar proteins FlaA1 and FlaB of porcine Serpulina species.

The periplasmic-flagellum (PF) proteins of Triton X-100-soluble and Triton X-100-insoluble sodium dodecyl sulfate-treated fractions from reference and field strains of Serpulina hyodysenteriae, Serpulina innocens, and Serpulina pilosicoli were characterized by Western blotting with a rabbit polyclonal antibody (PAb) specific for the 44-kDa PF sheath protein of S. hyodysenteriae (Z. Li, F. Dumas, D. Dubreuil, and M. Jacques, J. Bacteriol. 175:8000-8007, 1993) and a murine monoclonal antibody (MAb), designated 7G2, specific for the PF core FlaB proteins of S. hyodysenteriae. The MAb 7G2 reacted with a conserved epitope present in the 37-, 34-, and 32-kDa PF core FlaB proteins of all Serpulina species. This suggested that the core FlaB proteins are conserved among porcine Serpulina species. An immunoreactive band of approximately 44 kDa was present with all S. hyodysenteriae, S. innocens, and S. pilosicoli strains that were reacted with the PAb. The specificities of the PAb and the MAb for the FlaA1 and FlaB proteins of Serpulina species were confirmed by N-terminal amino acid sequencing of 44- and 37-kDa proteins, respectively, of S. hyodysenteriae and S. pilosicoli. Results from this study provide further evidence that the 44-kDa protein FlaA1 and the 37-, 34-, and 32-kDa FlaB proteins are conserved among porcine Serpulina species.

Identification of Serpulina species associated with porcine colitis by biochemical analysis and PCR.

A PCR system for the detection and identification of group IV spirochetes (Serpulina pilosicoli) was designed to complement biochemical tests, e.g., the hippurate hydrolysis and beta-glucosidase tests, and to verify the accuracy of a previously proposed biochemical classification system. The PCR assay was based on amplification of a segment of the 16S rRNA gene. Both primers were constructed to selectively amplify the 16S rRNA gene of Serpulina pilosicoli. All analyzed Serpulina strains exhibiting the capacity to hydrolyze hippurate and lacking beta-glucosidase activity, including the type strain for spirochetal diarrhea, P43, were amplified with the PCR system. All other tested strains, including type and field strains of different phenotypes of Serpulina species, as well as Salmonella species, Campylobacter species, and Escherichia coli strains, were negative in the assay. Among the tested strains were 18 Scottish field isolates originating from the mucosae of pigs with colitis. A simple classification scheme, suitable for routine classification of porcine intestinal spirochetes, is also presented. The scheme is based on hemolysis, indole production, and the hippurate hydrolysis test.

Purification and characterization of VSH-1, a generalized transducing bacteriophage of Serpulina hyodysenteriae.

Serpulina hyodysenteriae B204 cells treated with mitomycin (20 microg of mitomycin/ml of culture broth) lysed and released bacteriophages. Bacteriophage particles, precipitated by using polyethylene glycol and purified by CsC1 density gradient ultracentrifugation, had a buoyant density of 1.375 g/cm3 and consisted of a head (45-nm diameter) and an ultrastructurally simple (noncontractile) tail (64 by 9 nm) composed of at least 13 proteins with molecular masses ranging between 13 and 101 kDa. The purified bacteriophage has been designated VSH-1 (VSH for virus of S. hyodysenteriae). VSH-1 was incapable of lytic growth on any of five intestinal spirochete strains, representing three Serpulina species. VSH-1 nucleic acid was determined to be approximately 7.5 kb in size and to be linear, double-stranded DNA based on differential staining with acridine orange, DNase I sensitivity, electrophoretic mobility, and contour length as measured by electron microscopy. Phage DNA digested by the restriction enzymes SspI, AseI, EcoRV, and AflII gave electrophoretic banding patterns nearly identical to those of digested chromosomal DNA from S. hyodysenteriae. Additionally, VSH-1 DNA fragments hybridized with probes complementary to S. hyodysenteriae chromosomal genes nox and flaA1. When purified bacteriophages induced from cultures of S. hyodysenteriae A203 (deltaflaA1 593-762::cat) were added to growing cells of strain A216 (deltanox 438-760::kan), transductants (Cmr Kmr) were obtained at a frequency of 1.5 x l0(-6) per phage particle (enumerated by electron microscopy). These findings indicate that induced VSH-1 virions package DNA of S. hyodysenteriae and are capable of transferring host genes between cells of that spirochete. To our knowledge, this is the first report of genetic transduction of a spirochete.

Rapid identification of porcine Serpulina species by colony blot assay using a genus-specific monoclonal antibody.

An immunoglobulin G monoclonal antibody (mAb C9E8) recognising a genus-specific epitope on the 26 kDa protein of porcine Serpulina species organisms was used in a simple colony blot assay to detect Serpulina in cultures grown directly on blood agar plates from pig faeces and tissues. The mAb detected even a few colonies of the organism in the presence of an abundant growth of non-Serpulina organisms. The whole procedure was completed in less than three hours. A total of 123 strains of S hyodysenteriae and S innocens were correctly identified by the colony blot assay whereas all the 26 non-Serpulina Gram-negative organisms commonly isolated from faecal material or tissues of pigs remained negative. The assay was rapid, highly specific and sufficiently reliable to be used with confidence for identifying porcine Serpulina colonies directly on blood agar plates.

Prevalence of Serpulina species in relation to diarrhea and feed medication in pig-rearing herds in Sweden

Abstract Objective To determine prevalence of various pheno- and genotypes of Serpulina sp in young pigs in relation to diarrhea and feed medication in Swedish pig-rearing herds. Design Isolation of spirochetes. Phenotypical and genotypical classification. Sample Population Young pigs (n = 358) in 19 pig-rearing herds. Procedure Serpulina isolates were classified according to a biochemical scheme based on hemolysis, indole production, hippurate hydrolysis, and α-galactosidase, α-glucosidase, and β-glucosidase activities. The 16S rRNA sequences for 10 of the field strains and 2 type strains of Serpulina spp were aligned and compared. Minimum inhibitory concentrations of olaquindox for 9 of the strains were determined. Results Weakly β-hemolytic intestinal spirochetes (WBHIS) were isolated from 17 of the herds and 65% of the samples. More than 1 phenotype of WBHIS was found in 12 of the 19 herds. S hyodysenteriae was not isolated in any of the herds. Hippurate-positive WBHIS were isolated in 6 of 7 herds affected by diarrhea, but in only 1 of 8 herds without diarrhea. Hippurate-positive strains were closely related to the pathogenic strain P43 if judged from sequence comparisons. Strains with the same biochemical profile isolated within a herd had identical sequences, but when isolated from different herds, sequence differences were observed. The prevalence of WBHIS was reduced in herds medicated with olaquindox. Investigated field strains had minimum inhibitory concentration values ≤ 1 μg/ml for olaquindox. Conclusion The presence of WBHIS, with the ability to hydrolyze hippurate, was related to diarrhea in pig herds. Clinical Relevance Potentially pathogenic WBHIS can be distinguished from nonpathogenic strains by the hippurate hydrolysis test. (Am J Vet Res 1996;57:807–811)

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78 T, S. innocens strain B256 T, Brachyspira aalborgi strain 513A T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.

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.

Identification and characterization of genus-specific epitopes of Serpulina species using monoclonal antibodies

Four murine monoclonal antibodies (mAbs) designated as C9E8, A10, G12, and G8 which recognized both Serpulina hyodysenteriae and S. innocens were produced and characterized. The mAbs reacted with whole cell antigens in ELISA, indirect immunofluorescence and immunoblot assays. The mAbs did not show any cross reactivity in rapid dot ELISA or immunoblot assay with Leptospira icterohemorrhagiae, Campylobacter jejuni and Escherichia coli. Treatment of whole cell suspension with proteinase K and sodium periodate indicated that the reacting epitopes of the mAbs were protein in nature. The genus-specific antigens were identified as heat-stable proteins with molecular weight in the range of 26 to 45 kDa. Immunofluorescence and immunogold labelling studies showed that the antibody-binding epitopes were exposed on the outer-surface of the spirochaetal cell wall. The mAbs inhibited growth of reference strains of both S. hyodysenteriae and S. innocens in vitro but failed to cause agglutination. The detection of spirochaetal forms directly in fecal smears or paraffin-embeded tissue sections from experimentally infected pigs indicated that such mAbs were potentially useful for the diagnosis of swine spirochaetosis. This is the first report of mAbs identifying and characterizing common antigens of porcine Serpulina.