Evaluation of a 23S rDNA polymerase chain reaction assay for identification of Serpulina intermedia, and strain typing using pulsed-field gel electrophoresis.

A polymerase chain reaction assay, amplifying a 1027 base pair portion of the 23S rDNA gene, was evaluated for identification of the intestinal spirochaete Serpulina intermedia. A total of 34 strains of S. intermedia isolated from pigs and chickens and 195 strains of other related species were tested. The optimised assay correctly identified all the S. intermedia strains, but generated 11 false positive reactions, giving a test sensitivity of 100% and a test specificity of 94.3%. The false positive reactions were generated from strains of four different species of intestinal spirochaetes, and the product was of the original predicted size. This suggests that the primer sites selected on the 23S rRNA gene were not completely specific for S. intermedia. Pulsed-field gel electrophoresis was then developed to investigate diversity amongst the S. intermedia strains. All strains tested had distinct DNA banding patterns using Mlu1, although three isolates from chickens on the same farm appeared closely related. The collection exhibited considerable genetic diversity, and strains from pigs and chickens were distributed in clusters throughout the dendrogram produced. The most closely related porcine and avian strains shared only 62% similarity.

Analysis of Serpulina hyodysenteriae strain variation and its molecular epidemiology using pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was applied as a molecular typing tool for the spirochaete Serpulina hyodysenteriae, the agent of swine dysentery. Analysis of a collection of 40 mainly Australian isolates, previously characterized by other methods, divided these into 23 PFGE types. This confirmed that there are many strains of the spirochaete in Australia. PFGE was more discriminatory for strain typing than both multilocus enzyme electrophoresis and serotyping. It had similar discriminatory power to restriction endonuclease analysis, but the results of PFGE were easier to interpret. When applied to 29 isolates collected from 4 farms over periods of up to 8 years, 2 PFGE patterns were found on 3 farms, and a single pattern on the other. In each case a new strain had apparently emerged as a variant of an original parent strain. PFGE was found to be a powerful technique for investigating the molecular epidemiology of swine dysentery outbreaks.

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.

Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis.

Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae, the agent of swine dysentery, and S. pilosicoli, the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 10(3)-10(4) cells of either organism seeded into 0.2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli, both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation.

Genetic relatedness amongst intestinal spirochaetes isolated from rats and birds.

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal spirochaetes (Serpulina spp.) isolated from rats (17), rheas (7), chickens, (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for porcine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain porcine strains of Serpulina hyodysenteriae. The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.

Pulsed-field gel electrophoresis for sub-specific differentiation of Serpulina pilosicoli (formerly 'Anguillina coli').

Pulsed-field gel electrophoresis (PFGE) was developed for subspecific differentiation of Serpulina pilosicoli, and was applied to 52 isolates recovered from cases of intestinal spirochaetosis (IS) in pigs, dogs, human beings and various avian species. The technique was highly sensitive, differentiating the isolates into 40 groupings. Only six groups contained more than one isolate; in five of these groups isolates with the same banding pattern were either from pigs in the same herds (four groups), or from humans in the same community: the sixth group contained two identical Australian porcine isolates from unrelated herds in different states. Overall S. pilosicoli isolates were genetically diverse, but in some cases isolates cultured from the same or different animal species were closely related. This suggested the likelihood of cross-species transmission, including zoonotic spread. PFGE was a powerful tool for epidemiological studies of S. pilosicoli and also allowed examination of genetic relationships between isolates.

Distribution of the smpA gene from Serpulina hyodysenteriae among intestinal spirochaetes.

Forty intestinal spirochaete strains were investigated for nucleotide sequences related to the smpA locus from Serpulina hyodysenteriae by Southern hybridization of chromosomal DNA using the smpA locus from S. hyodysenteriae strain P18A as a probe and by PCR using primers internal to the smpA gene. The intensity of the hybridization signal at high stringency and positive PCR results suggested that 12 S. hyodysenteriae strains possessed a similar nucleotide sequence. PCR was negative for another 12 S. hyodysenteriae strains and the hybridization signal obtained from 11 of these was weak and one was negative. All S. hyodysenteriae strains hybridized under low stringency conditions. These results indicated that there is variation among the smpA loci of S. hyodysenteriae strains. Among seven strains of S. innocens, and the proposed species 'S. intermedius' and 'S. murdochii', hybridization was weak and no PCR products were obtained, suggesting that these species have sequences related to, but divergent from, the smpA sequences of strains of S. hyodysenteriae. Both gene probe hybridization and PCR analysis of nine strains of the proposed new genus 'Anguillina', including isolates from pigs and humans, gave negative results.

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis.

A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name 'Anguillina coli'. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for 'A. coli'. The PCR was used to correctly identify DNA extracted from 43 isolates of 'A. coli' from humans and pigs, whilst no product was produced from Escherichia coli, or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi. The amplification provided a rapid and simple means of identifying DNA from isolates of 'A. coli', and could be used on boiled whole 'A. coli' cells, with a detection limit equivalent to 2.5 x 10(2) cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.

Clonal analysis and virulence of Australian isolates of Streptococcus suis type 2.

Multilocus enzyme electrophoresis was used to divide 124 Australian isolates of Streptococcus suis type 2 into 17 electrophoretic types (ETs). Isolates in ET 1 were the most frequent cause of disease amongst Western Australian pigs, but isolates of ET 8 were more commonly associated with disease in other Australian states. Multiple isolates from 10 of 19 farms all belonged to the same ET, whilst isolates from the other farms belonged to between 2 and 4 different ETs. Some isolates could be differentiated further by DNA restriction endonuclease analysis, whilst others with the same restriction pattern were located in different, but closely-related ETs. Fourteen isolates were tested for their virulence in mice. Most caused disease if given in high numbers, but isolates in ET 1 were virulent at lower dose rates. This virulent clone also was distinguished by the fact that 80% of isolates produced extracellular factor (EF).