Diagnosis of swine dysentry and intestinal spirochaetosis by the use of polymerase chain reaction tests on faecal samples

The polymerase chain reaction (PCR) is a technique based on amplification of specific DNA sequences/ the products of which may be detected using electrophoresis (Saiki et al., 1985). The DNA sequences from Serpulina hyodysenteriae, the causative agent of swine dysentery (SD) (Combs and Hampson/ 1992), and from the spirochaete previously called "Anguillina coli" (Lee et al., 1993), which is associated with intestinal spirochaetosis (IS) (Park et al., 1995), were used to design PCR tests, and these were applied to the detection of these bacteria in pigs

Pulsed field gel electrophoresis analysis of Serpulina hyodysenteriae isolates

Molecular typing techniques have been used to determine the extent of strain variation in Serpulina hyodysenteriae, and have been useful in studying the molecular epidemiology of swine dysentery (SD). Techniques used have included multilocus enzyme electrophoresis (MEE) (6), DNA restriction endonuclease analysis (REA) (3), restriction fragment length polymorphism (RFLP) analysis (5), and random amplification of polymorphic DNA (RAPD) analysis (4). In the current study, pulsed field gel electrophoresis (PFGE) was developed as a simple and reliable alternative method for subspecific differentiation of the species

The use of polymerase chain reaction for the identification of Serpulina hyodysenteriae

The isolation and identification of Serpulina hyodysenteriae is considered necessary to obtain a definitive diagnosis of swine dysentery (1). Identification has traditionally been carried out using phenotypic tests, although recently DNA hybridisation tests have been reported as an alternative (2). The usefulness of these and other tests relies on their ability to distinguish between S. hyodysenteriae and other spirochaetes that are frequently isolated from the porcine colon (3). Identification of pathogenic bacteria has been successfully carried out using the polymerase chain reaction ( PRC) ( 4), and in the current study this method was investigated as a means of identifying S. hyodysenteriae

PCR for identification of spirochaetes associated with intestinal spirochaetosis

Weakly haemolytic intestinal spirochaetes, distinct from both Serpulina hyodysenteriae and Serpulina innocens, have been reported to cause a disease called intestinal spirochaetosis, or spirochaetal diarrhoea, in weaned pigs (1, 2, 3, 4) . Infected animals develop a sloppy mucoid diarrhoea and a reduction in growth rate. These spirochaetes are slightly more slender than S. innocens, and characteristically only have 4-6 axial flagellae ( 5). Strain P43/6/78, which was isolated from the first recorded case of porcine intestinal spirochaetosis ( 1), has been deposited with the American Type Culture Collection (as ATCC 51139) as the type strain of a possible new genus and species of spirochaete ( "Anguillina coli") ( 5). In the current study we have used a polymerase chain reaction (PCR) amplification of a section of the region encoding the 16s ribosomal RNA gene as a rapid and specific means of identifying these bacteria

Polymerase chain reaction targetting the nox gene for identification of Serpulina intermedia in pigs

Serpulina intermedia (formerly "S. intemedius") is a recently named species of weakly B-haemolytic, indole positive anaerobic intestinal spirochaete (7). In diagnostic laboratories it can be easily confused with the strongly β-haemolytic, indole positive S. hyodysenteriae (the agent of swine dysentery), or one of several other weakly β-haemolytic non-pathogenic Serpulina species. S. intermedia is considered to be a pathogen of poultry (5), however evidence for its pathogenic potential in pigs remains equivocal (3). Strains of what appear to be S. intermedia have been isolated from pigs with diarrhoea in Poland (1) and Sweden (2), but in experimental studies infection of conventional pigs with S. intermedia type strain PWS/ A did not result in disease (4). The purpose of this study was to develop a polymerase chain reaction (PCR) test for the identification of S. intermedia strains using sequence information derived from the NADH oxidase (nox) gene of the spirochaete. NADH oxidase has been detected in every Serpulina strain tested and thus may be an identifying trait for the genus (7)

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

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 103-104 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

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

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