Identification of porcine intestinal spirochetes by PCR-restriction fragment length polymorphism analysis of ribosomal DNA encoding 23S rRNA

The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.     

SmpB: a novel outer membrane protein present in some Brachyspira hyodysenteriae strains

A novel outer membrane protein-encoding gene was identified in Brachyspira hyodysenteriae. The predicted protein, SmpB, was encoded by a gene that contains regions of identity with that encoding the previously identified lipoprotein SmpA. However, the majority of the reading frame encoding SmpA and SmpB share no detectable similarity. Analysis of several strains revealed that B. hyodysenteriae harbours either smpA or the newly identified gene smpB, but not both. smpB encodes for a slightly larger protein than smpA, 17.6 and 16.8 kDa, respectively. The predicted proteins share an identical leader sequence and the first 10 amino acids of the mature protein, however, the remainder of the predicted protein sequence shows no similarity. It is hypothesised that smpA and smpB are present on the same area of the chromosome. The proteins are antigenically unique, as antisera raised against a strain of B. hyodysenteriae that expresses SmpA cannot detect SmpB and vice versa. Although the presence of an identical leader peptide suggests identical localisation of SmpA and SmpB, it is not known if the two predicted proteins share similar function.     

Faecal excretion of intestinal spirochaetes by urban dogs, and their pathogenicity in a chick model of intestinal spirochaetosis

This study aimed to obtain information about the types of spirochaetes colonising urban dogs in Thailand, and to investigate their pathogenic potential in a day-old chick model of intestinal spirochaetosis. Spirochaetes were isolated from the faeces of six of 47 (12.8%) healthy dogs and 11 of 104 (10.6%) dogs with diarrhoea. Their biochemical properties and 16S ribosomal DNA sequences were analysed. Four isolates were identified as Brachyspira pilosicoli, three resembled "Brachyspira pulli", nine clustered with "Brachyspira canis" and one was similar to Brachyspira intermedia. Canine isolates of B. pilosicoli, "B. canis" and "B. pulli", and control strains of Brachyspira hyodysenteriae, B. pilosicoli and Brachyspira innocens colonised experimentally infected day-old chicks. The chicks did not develop diarrhoea, but were significantly lighter than the non-infected group and those infected with B. innocens after 21 days (P<0.05). Using immunohistochemistry, spirochaetes were observed covering the surface epithelium and in the crypts of chicks in all three groups challenged with the canine isolates. Variable histopathological changes were seen, with the greatest inflammatory cell infiltration into the lamina propria occurring in the group infected with "B. pulli". Canine "B. canis", "B. pulli" and B. pilosicoli isolates may have pathogenic potential.     

Diagnosis of swine dysentery and spirochaetal diarrhea. III. Results of cultural and biochemical differentiation of intestinal Brachyspira species by routine culture from 1997 to 1999

A survey is given on the occurrence and distribution of different Brachyspira species in pigs, in the northwest of Germany. In total 2975 specimen (feces, fecal swabs, colon) were taken and sent for laboratory analysis during the years 1997 to 1999. 1218 Brachyspira (B.) strains were found by cultural analysis. 1757 samples (59%) were negative. The cultural and biochemical differentiation revealed 720 (59.1%) strains B. hyodysenteriae (77.5% were indole negative), 22 (1.8%) B. pilosicoli, 29 (2.4%) B. intermedia, 167 (3.7%) B. innocens and 114 (9.4%) B. murdochii. 166 (13.6%) strains could not be identified. These strains could either not be compared with any of the described species by the methods used or it was impossible to achieve a pure culture from these isolates. The results demonstrate the wide spread of B. hyodysenteriae in pig herds in the northwest of Germany with a very high prevalence of indole negative strains. The most frequent strain was B. hyodysenteriae. B. pilosicoli which causes spirochaetal diarrhoea was rarely isolated and seems not to play an important role in Germany. Experience from routine cultures for Brachyspira give evidence that it is more useful to examine faeces from single pigs instead of pooled samples from a herd. It is recommended to use special transport media for the transport of the specimen.     

Identification of genes associated with prophage-like gene transfer agents in the pathogenic intestinal spirochaetes Brachyspira hyodysenteriae, Brachyspira pilosicoli and Brachyspira intermedia

VSH-1 is an unusual prophage-like gene transfer agent (GTA) that has been described in the intestinal spirochaete Brachyspira hyodysenteriae. The GTA does not self-propagate, but it assembles into a virus-like particle and transfers random 7.5kb fragments of host DNA to other B. hyodysenteriae cells. To date the GTA VSH-1 has only been analysed in B. hyodysenteriae strain B204, in which 11 late function genes encoding prophage capsid, tail and lysis elements have been described. The aim of the current study was to look for these 11 genes in the near-complete genomes of B. hyodysenteriae WA1, B. pilosicoli 95/1000 and B. intermedia HB60. All 11 genes were found in the three new strains. The GTA genes in WA1 and 95/1000 were contiguous, whilst some of those in HB60 were not-although in all three strains some gene rearrangements were present. A new predicted open reading frame with potential functional importance was found in a consistent position associated with all four GTAs, located between the genes for head protein Hvp24 and tail protein Hvp53, overlapping with the hvp24 sequence. Differences in the nucleotide and predicted amino acid sequences of the GTA genes in the spirochaete strains were consistent with the overall genetic distances between the strains. Hence the GTAs in the two B. hyodysenteriae strains were considered to be strain specific variants, and were designated GTA/Bh-B204 and GTA/Bh-WA1 respectively. The GTAs in the strains of B. intermedia and B. pilosicoli were designated GTA/Bint-HB60 and GTA/Bp-95/1000 respectively. Further work is required to determine the extent to which these GTAs can transfer host genes between different Brachyspira species and strains.     

A possibility of application of the 105-kilodaltons protein of Brachyspira alvinipulli cross-reacting with antisera to five species in the genus Brachyspira to diagnosis

The antigenic properties of Brachyspira (B.) alvinipulli ATCC 51933 and strain C2 were analyzed and compared with those of B. hyodysenteriae ATCC 27164 and ATCC 31212, B. pilosicoli ATCC 51139, B. innocens ATCC 29796 and B. aalborgi NCTC 11492. In gel immunodiffusion tests, a protein in B. alvinipulli ATCC 51933 reacted strongly with anti-B. alvinipulli ATCC 51933-serum and formed two precipitin lines. Furthermore, by an immunoblotting technique, the 105-kilodaltons (kDa) protein in B. alvinipulli ATCC 51933 reacted strongly with each of the antisera to B. hyodysenteriae, B. pilosicoli, B. innocens and B. aalborgi. Therefore, the 105-kDa protein could be applied to diagnosis of chicken infection by B. alvinipulli and B. pilosicoli. But the 105-kDa protein reacting with the anti-B. alvinipulli ATCC 51933-serum was not confirmed in B. hyodysenteriae, B. pilosicoli, B. innocens and B. aalborgi. The N-terminal amino acid sequence of the 105-kDa protein isolated from B. alvinipulli ATCC 51933 was Met-Lys-Lys-Met-Val-Tyr-Phe-Phe-Gly-Asn. The amino acid alignment of this protein possessed 50% homology with the periplasmic-iron-binding protein BitC in B. hyodysenteriae.     

Diagnosis of swine dysentery and spirochaetal diarrhea. 2. Effort of macrorestriction analysis for differentiation of intestinal Serpulina relative to determination by culture-biochemical markers following species classification]

Genotypic differentiation by means of macrorestriction fragment profile analysis using Mlul restriction enzyme was carried out differentiating 41 Serpulina field strains from swine (38), dog (2) and a rat as well as ten type and reference strains into 40 electrophoretic types. A dendrogram was created using the average linkage between groups method. At a level of 50% similarity the patterns could be divided into six groups that roughly corresponded to the results yielded by cultural and biochemical methods formerly (FELTRUP et al. 1999). Five of these clusters corresponded to the five known porcine Serpulina species, one cluster contained the S. pilosicoli isolates from dog and rat included in this study. Interestingly all nine investigated indole negative, strongly haemolytic isolates were clustered together in one group with the S. hyodysenteriae strains, so that incidence of indole negative variants of S. hyodysenteriae was confirmed. Because of being grouped together with two S. intermedia isolates, the suitability of B 256 as S. innocens type strain is--in accord to investigations carried out by PETTERSSON et al (1996)--called in question.     

Assessment of diagnostics and antimicrobial susceptibility testing of Brachyspira species using a ring test

There is no ring test for quality assessment available in Europe for diagnostics and antimicrobial susceptibility testing of the fastidious, anaerobic bacteria of the genus Brachyspira. Therefore, an international ring test for Brachyspira spp. was performed once a year during 2002-2004. Two sets of coded samples were prepared and distributed on each occasion. One set comprised six swabs dipped in pig faeces spiked with Brachyspira spp. intended for diagnostics. The other set comprised two pure strains intended only for susceptibility testing. All methods used were in-house methods. The species used were Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira innocens, Brachyspira murdochii and Brachyspira intermedia. In most cases, the correct Brachyspira spp. were detected. However, the results showed that Brachyspira spp. could be difficult to identify, especially if two Brachyspira spp. were mixed or if the concentration of Brachyspira in faeces was low. Additionally, some laboratories reported Brachyspira growth in control samples that were not seeded with any spirochaetes. The lowest detection level was 10(2) bacteria/ml faeces for both B. hyodysenteriae and B. pilosicoli. The susceptibility tests performed showed that disc diffusion was not recommendable for Brachyspira spp. Extended antimicrobial dilution series gave most congruent results. The diversity of the results highlights the importance of ring tests for a high quality of diagnostics and antimicrobial susceptibility tests for Brachyspira spp. This is the first ring test described for Brachyspira spp.     

Demonstration of genes encoding virulence and virulence life-style factors in Brachyspira spp. isolates from pigs

The distribution of many genes encoding virulence and virulence life-style (VL-S) factors in Brachyspira (B.) hyodysenteriae and other Brachyspira species are largely unknown. Their knowledge is essential e.g. for the improvement of diagnostic methods targeting the detection and differentiation of the species. Thus 121 German Brachyspira field isolates from diarrhoeic pigs were characterized down to the species level by restriction fragment length polymorphism analysis of the nox gene and subsequently subjected to polymerase chain reaction detecting VL-S genes for inner (clpX) and outer membrane proteins (OMPs: bhlp16, bhlp17.6, bhlp29.7, bhmp39f, bhmp39h), hemolysins (hlyA/ACP, tlyA), iron metabolism (ftnA, bitC), and aerotolerance (nox). For comparison, B. hyodysenteriae reference strains from the USA (n=7) and Australia (2) were used. Of all genes tested only nox was detected in all isolates. The simultaneous presence of both the tlyA and hlyA/ACP was restricted to the species B. hyodysenteriae. The hlyA infrequently occurred also in weakly hemolytic Brachyspira. Similarly to tlyA and hlyA all B. hyodysenteriae strains contained the ferritin gene ftnA which was also found in two Brachyspira intermedia isolates. OMP encoding genes were present in B. hyodysenteriae field isolates in rates of 0% (bhlp17.6, bhmp39h), 58.1% (bhlp29.7), and 97.3% (bhmp39f). Since the study revealed a high genetic heterogeneity among German B. hyodysenteriae field isolates differentiating them from USA as well as Australian strains, targets for diagnostic PCR were limited to the nox gene (genus specific PCR) as well as to the species specific nox(hyo) gene and the combination of hlyA and tlyA which allow to specifically detect B. hyodysenteriae.     

Occurrence of Lawsonia Intracellularis and Brachyspira spp. infection in swine suffering from diarrhoea

Principal aim of this study was to examine fecal samples from pigs suffering from diarrhea for the presence of Lawsonia intracellularis, Brachyspira hyodysenteriae and Brachyspira pilosicoli. The molecular techniques such as PCR and nested PCR were employed to detect the presence of p78 fragment of genomic DNA specific for Lawsonia intracellularis as well as fragment of tlyA gene specific for Brachyspira hyodysenteriae and 16S rDNA gene of Brachyspira pilosicoli. We assumed that about 25% of pigs were infected with Lawsonia intracellularis, about 10% with Brachyspira hyodysenteriae and only 0,8% with Brachyspira pilosicoli. In about 3% mixed infection with L. intracellularis and B. hyodysenteriae was observed. Results were comparable in herds that differed in quantity, breeding technology, hygienic standards and preventive treatment with different chemotherapeutics.     

Amplification and sequencing of Brachyspira spp. specific portions of nox using paraffin-embedded tissue samples from clinical colitis in Austrian pigs shows frequent solitary presence of Brachyspiramurdochii

Brachyspira infections are significant causes of enterocolitis in pigs. In order to differentiate pathogenic species (Brachyspira (Br.) hyodysenteriae, Brachyspira pilosicoli) from less pathogenic or non-pathogenic species (Brachyspira intermedia, Brachyspira innocens, Brachyspira murdochii) in paraffin-embedded tissue samples a polymerase chain reaction (PCR) protocol allowing identification of Brachyspira at species level in archival material was developed. This approach was complemented by sequencing of the PCR amplification products. All seven cases presented with clinical and morphological Brachyspira-associated enterocolitis. Br. hyodysenteriae was not identified in any of the cases, while Br. pilosicoli was identified in a single case in conjunction with Br. murdochii. One case each was found positive for Br. innocens and Br. intermedia. Interestingly, the majority of cases presented as single or double infections with Br. murdochii. In some of the pigs other pathogens, like porcine circovirus-2 or Lawsonia intracellularis were present. These observations point at the possibility that under certain conditions even Brachyspira species of low pathogenicity can multiplicate extensively and lead to Brachyspira-associated enterocolitis.     

Electron microscopic studies on the interaction between normal mice peritoneal phagocytes and Treponema hyodysenteriae, Treponema innocens and Bacteroides vulgatus.

One hundred and twenty female mice (CF1 strain) were divided into three groups of 40. The first group was injected intraperitoneally with broth cultures of Treponema hyodysenteriae. The second group was injected with a combination of T. hyodysenteriae and Bacteroides vulgatus. The third group was injected with Treponema innocens. Peritoneal wash from four mice of each group was collected at eight time intervals postinjection, then prepared for and examined by light and electron microscopy. Peritoneal wash from one mouse at each time interval was prepared for microbiological examination. Treponema hyodysenteriae produced peritoneal macrophage aggregation, transient neutrophilia and macrophage cytolysis. Cytolysis was characterized by rarefaction of the cytoplasm, vesiculation of the endoplasmic reticulum, mild swelling of the mitochondria and disruption of the nuclear and ctyoplasmic membranes. The combination of T. hyodysenteriae and B. vulgatus produced macrophage aggregation and marked neutrophil necrosis. Peritoneal macrophages phagocytized more T. hyodysenteriae than B. vulgatus during early postinjection intervals. Treponema innocens failed to produce cytotoxicity of peritoneal macrophages but did produce macrophage aggregation and transient neutrophilia. Treponema hyodysenteriae and T. innocens did not multiply in the mice peritoneal cavity and were reisolated up to 16 hours postinjection. Bacteroides vulgatus was reisolated up to 24 hours postinjection.     

Protection of pigs from swine dysentery by vaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0.047) than for the control groups, with a approximately 50% reduction. BmpB appears to have potential as an SD vaccine component.     

Comparative evaluation of molecular assays for the identification of intestinal spirochaetes from diseased pigs

Rapid identification of porcine Brachyspira species is required in order to differentiate pathogenic from non-pathogenic species. The aim of our study was to compare a recently described genetic method based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP), nox RFLP-PCR assay, and three species-specific PCRs described previously in the literature with a 16S rRNA gene RFLP-PCR discriminatory reference assay (16S RFLP-PCR) for the identification of Brachyspira spp. of swine origin. In this study, 20 porcine spirochaetal strains were identified and compared to 33 reference strains by 16S RFLP-PCR and nox RFLP-PCR and three species-specific PCRs. RFLP-PCR methods showed concordant results for 47 strains and discordances for 6 strains (2 differently identified and 4 not revealed by nox RFLP-PCR). In our hands species-specific PCRs showed concordant results with 16S and nox RFLP-PCR for 43 strains and discordances for 10 strains (2 differently identified and 8 not amplified). The same results observed testing the 20 field-isolated spirochaetes were obtained for the corresponding porcine faecal samples. The detection limit was 10(2) -10(3) cells/g of faeces for 16S rRNA gene PCR and 10(4) cells/g of faeces for nox PCR. In our experience nox RFLP-PCR appeared successful for the speciation of B. hyodysenteriae reserving 16S RFLP-PCR for all other pathogenic and non-pathogenic Brachyspira species. Among the species-specific PCR assays tested only that for B. pilosicoli was useful in our hands.     

The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes

The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.     

Detection of Brachyspira hyodysenteriae, Lawsonia intracellularis and Brachyspira pilosicoli in feral pigs

Feral pigs are recognized as being a potential reservoir of pathogenic microorganisms that can infect domestic pigs and other species. The aim of this study was to investigate whether feral pigs in Western Australia were colonized by the pathogenic enteric bacteria Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli. A total of 222 feral pigs from three study-populations were sampled. DNA was extracted from faeces or colonic contents and subjected to a previously described multiplex PCR for the three pathogenic bacterial species. A subset of 61 samples was cultured for Brachyspira species. A total of 42 (18.9%) of the 222 samples were PCR positive for L. intracellularis, 18 (8.1%) for B. hyodysenteriae and 1 (0.45%) for B. pilosicoli. Four samples were positive for both L. intracellularis and B. hyodysenteriae. Samples positive for the latter two pathogens were found in pigs from all three study-sites. A strongly haemolytic B. hyodysenteriae isolate was recovered from one of the 61 cultured samples. Comparison of a 1250-base pair region of the 16S rRNA gene amplified from DNA extracted from the isolate and five of the B. hyodysenteriae PCR positive faecal samples helped confirm these as being from B. hyodysenteriae. This is the first time that B. hyodysenteriae has been detected in feral pigs. As these animals range over considerable distances, they present a potential source of B. hyodysenteriae for any domesticated pigs with which they may come into contact.     

The prevalences of Brachyspira spp. and Lawsonia intracellularis in Swedish piglet producing herds and wild boar population

The aim of the present study was to survey the prevalences of the enteric pathogens Brachyspira hyodysenteriae, Brachyspira pilosicoli and Lawsonia intracellularis in Swedish growing pigs and in the Swedish wild boar population and to relate these findings to clinical signs. The study included 105 randomly selected herds, constituting approximately one third of Swedish herds with a herd size of >100 sows. The herds were located all over the country. In these herds, growth promoters were not used and pigs sampled were not subjected to any medication. From each herd, samples were taken from 10 growing pigs aged 8-12 weeks, corresponding to approximately 2.5% of all growing pigs present in the herd at the sampling occasion. If possible, the samples were taken from pigs with diarrhoea. Forty-eight faecal samples and 71 rectal swabs were also taken from free-living wild boars (31 piglets, 19 growers and 21 adult animals) at shooting. The samples were analysed by culture and biochemical tests for the presence of Brachyspira spp. and by nested PCR for the presence of L. intracellularis. Brachyspira hyodysenteriae was not demonstrated in any sample. Brachyspira intermedia was detected in 22 samples originating from 15 herds, Brachyspira innocens/Brachyspira murdochii was detected in 370 samples from 82 herds and B. pilosicoli was detected in 134 samples originating from 34 herds. In 21 herds and in 534 samples, no Brachyspira spp. were detected. Lawsonia intracellularis was demonstrated in 285 samples from 50 herds. Further, 418 samples from conventional herds were negative with respect to L. intracellularis and in 345 samples the PCR had been inhibited. All samples from the wild boars were negative for Brachyspira spp., 12 of 48 samples were negative for L. intracellularis, and in 36 wild boar samples, the PCR was inhibited.     

Evidence that the 36 kb plasmid of Brachyspira hyodysenteriae contributes to virulence

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently the genome of virulent Australian B. hyodysenteriae strain WA1 was sequenced, and a 36 kilobase (kb) circular plasmid was identified. The plasmid contained 31 genes including six rfb genes that were predicted to be involved with rhamnose biosynthesis, and others associated with glycosylation. In the current study a set of PCRs was developed to amplify portions of nine of the plasmid genes. When used with DNA extracted from virulent strain B204, PCR products were generated, but no products were generated with DNA from avirulent strain A1. Analysis of the DNA using pulsed field gel electrophoresis (PFGE) identified a plasmid band in strains WA1 and B204, but not in strain A1. These results demonstrate that strain A1 does not contain the plasmid, and suggests that lack of the plasmid may explain why this strain is avirulent. To determine how commonly strains lacking plasmids occur, DNA was extracted from 264 Australian field isolates of B. hyodysenteriae and subjected to PCRs for three of the plasmid genes. Only one isolate (WA400) that lacked the plasmid was identified, and this absence was confirmed by PFGE analysis of DNA from the isolate and further PCR testing. To assess its virulence, 24 pigs were experimentally challenged with cultures of WA400, and 12 control pigs were challenged with virulent strain WA1 under the same conditions. Significantly fewer (P=0.03) of the pigs challenged with WA400 became colonised and developed SD (13/24; 54%) compared to the pigs infected with WA1 (11/12; 92%). Gross lesions in the pigs colonised with WA400 tended to be less extensive than those in pigs colonised with WA1, although there were no obvious differences at the microscopic level. The results support the likelihood that plasmid-encoded genes of B. hyodysenteriae are involved in colonisation and/or disease expression.     

Detectability and prevalence of Brachyspira species in herds rearing health class feeder pigs in Finland

Faeces samples were taken three times at two-week intervals, from the farrowing units of four herds of known Brachyspira (formerly Serpulina) status and one of unknown Brachyspira status. Brachyspira hyodysenteriae, Brachyspira pilosicoli, Brachyspira intermedia and Brachyspira group III were isolated from the faecal samples from the weaners in the herds using either a maximum of 50 ppm of olaquindox or no feed additives. The detection rates were relatively consistent. However, B hyodysenteriae was not detected at one sampling in a known positive herd. The prevalence of Brachyspira species was also studied in feeder pigs originating from LSO 2000 health class farrowing units, comparable with specific pathogen-free herds. These farms were free from swine dysentery, sarcoptic mange, swine enzootic pneumonia and progressive atrophic rhinitis. Fifty of 428 herds were sampled once. B hyodysenteriae was not isolated from any of them, but B intermedia, B pilosicoli and Brachyspira group III were isolated from five, 14 and 37 of the herds, respectively. The detection of Brachyspira species did not relate to the prevalence of diarrhoea in the herds, as judged by the farmers. The herds using carbadox (40 to 50 ppm) had a lower prevalence of Brachyspira species than those using olaquindox (40 to 50 ppm).