Intestinal Spirochaete Brachyspira Research Articles

An atypical weakly haemolytic strain of Brachyspira hyodysenteriae is avirulent and can be used to protect pigs from developing swine dysentery

The anaerobic intestinal spirochaete Brachyspira hyodysenteriae colonises the large intestine of pigs and causes swine dysentery (SD), a severe mucohaemorrhagic colitis. SD occurs worldwide, and control is hampered by a lack of vaccines and increasing antimicrobial resistance. B. hyodysenteriae strains typically produce strong beta-haemolysis on blood agar, and the haemolytic activity is thought to contribute to the pathogenesis of SD. Recently, weakly haemolytic variants of B. hyodysenteriae have been identified in Europe and Australia, and weakly haemolytic strain D28 from Belgium failed to cause disease when used experimentally to infect pigs. Moreover, pigs colonised with D28 and then challenged with virulent strongly haemolytic strain B204 showed a delay of 2–4 days in developing SD compared to pigs not exposed to D28. The current study aimed to determine whether Australian weakly haemolytic B. hyodysenteriae strain MU1, which is genetically distinct from D28, could cause disease and whether exposure to it protected pigs from subsequent challenge with strongly haemolytic virulent strains. Three experimental infection studies were undertaken in which no diseases occurred in 34 pigs inoculated with MU1, although mild superficial lesions were found in the colon in 2 pigs in one experiment. In two experiments, significantly fewer pigs exposed to MU1 and then challenged with strongly haemolytic virulent strains of B. hyodysenteriae developed SD compared to control pigs not previously exposed to MU1 (p = 0.009 and p = 0.0006). These data indicate that MU1 lacks virulence and has potential to be used to help protect pigs from SD.

Vaccination of chickens with the 34 kDa carboxy-terminus of Bpmp72 reduces colonization with Brachyspira pilosicoli following experimental infection

ABSTRACTThe anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of a variety of species of mammals and birds, and may result in colitis, diarrhoea and reductions in growth rate. Naturally occurring infections in chickens are largely confined to adult laying and breeding birds. In this study, the 34 kD carboxy-terminus of the prominent outer membrane protein Bmp72 of B. pilosicoli was expressed as a histidine-tagged recombinant protein and used to immunize two groups (B and C) of 15 individually housed layer chickens. Vaccination was with either 100 μg (B) or 1 mg (C) protein emulsified with Freund’s incomplete adjuvant delivered into the pectoral muscles, followed three weeks later by 1 mg of protein in phosphate buffered saline delivered via crop tube. Two weeks later these and 15 non-vaccinated positive control birds (group A) housed in the same room were challenged via crop tube with B. pilosicoli avian strain CPS1. B. pilosicoli was detected in the faeces of all control birds and in 14 of the vaccinated birds in each vaccinated group at some point over the 30-day period following challenge. Colonization was delayed and the duration of excretion was significantly reduced (P = 0.0001) in both groups of vaccinated birds compared to the non-vaccinated control birds. Fewer immunized birds had abnormal caecal contents at post mortem examination compared to non-vaccinated birds, but the difference was not statistically significant. This study indicates that recombinant Bmp72 C-terminus has potential to be developed for use as a vaccine component to provide protection against B. pilosicoli infections.RESEARCH HIGHLIGHTSLaying chickens were immunized with recombinant Brachyspira pilosicoli membrane protein Bpmp72.Immunized birds had a highly significant reduction in the duration of colonization.Fewer immunized than control birds had abnormal caecal contents after infection.Bpmp72 showed potential for use as a novel vaccine component for B. pilosicoli.

Occurrence of dysentery-like diarrhoea associated with Brachyspira suanatina infection on a German fattening pig farm

The anaerobic intestinal spirochaete Brachyspira (B.) suanatina was first described in 2007 but since then no further isolates have been reported from pigs. Accordingly, when the species was validly published...

Brachyspira hyodysenteriae isolated from apparently healthy pig herds following an evaluation of a prototype commercial serological ELISA

Swine dysentery (SD) is a disease mainly of grower/finisher pigs characterised by severe mucohaemorrhagic colitis. The classical aetiological agent is the anaerobic intestinal spirochaete Brachyspira hyodysenteriae, although “Brachyspira hampsonii” and Brachyspira suanatina also cause SD. This study reports on the unexpected isolation of B. hyodysenteriae from pigs in apparently healthy herds that gave positive reactions when tested with a prototype commercial serological ELISA for detecting herds infected with B. hyodysenteriae (Priocheck®Brachyspira porcine Ab ELISA). The ELISA was tested with sera collected at abattoirs from 1770 slaughtered pigs from 30 Australian herds, including 12 with a history of SD and18 that were considered by their consulting veterinarians to be healthy. The latter herds had no history of SD and did not routinely use antimicrobials that may have masked the disease. Based on the recommended ELISA cut-off value, 25 herds were recorded as showing evidence of infection, including 11 of 12 herds that were considered infected by the submitters and 14 of the 18 “healthy” herds. When faecal or colonic wall samples from 11 of the 14 “false positive” herds subsequently were culturing 6–24 months after the original ELISA testing was completed, different strains of B. hyodysenteriae were isolated from six herds, including a high-health status breeding herd. The existence of apparently healthy herds that are colonised by B. hyodysenteriae has major implications for the control of SD. Had the ELISA not been trialled it is unlikely that colonic samples from these herds would have been cultured and the colonisation identified.

Release and Cytokine Production of BmpB from BmpB-Loaded pH-Sensitive and Mucoadhesive Thiolated Eudragit Microspheres.

Swine dysentery is a contagious mucohaemorrhagic colitis of pigs that is caused by anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Recently, an outer membrane lipoprotein of B. hyodysenteriae (BmpB) has been identified, and the mice or pigs immunized with a recombinant BmpB generated antibodies recognizing the native BmpB of B. hyodysenteriae. In this study, we cloned, expressed and purified BmpB protein from E. coli and used it as a vaccine candidate for oral delivery. The BmpB was encapsulated into the pH-sensitive and thiolated Eudragit microspheres (TEMS). The sizes of the microspheres ranged from 5-20 μ. About 22-34% of BmpB were released from the BmpB-loaded TEMS within 24 h at stomach pH 2.0 whereas the release of BmpB from the BmpB-loaded TEMS was 35% in the first one hour and reached 81% within 24 h at intestinal pH 7.2. These data revealed that the BmpB could be protected in the harsh gastric condition. Mucoadhesive experiment in vitro showed that TEMS have high binding affinity with the mucin glycoproteins of porcine intestine. Finally, in vitro production of cytokines from immune cells treated with the BmpB-loaded TEMS suggested that the TEMS would be a promising approach for oral delivery of BmpB as vaccine candidate.

Absence of a set of plasmid-encoded genes is predictive of reduced pathogenic potential in Brachyspira hyodysenteriae.

The gene content of 14 strains of the intestinal spirochaete Brachyspira hyodysenteriae was compared using a DNA microarray. A consistent difference occurred in a block of four genes on the ~36 Kb plasmid, with these being present in six virulent strains and absent in eight strains with reduced pathogenic potential. These genes encoded a predicted radical S-adenosylmethionine domain protein, a glycosyl transferase group 1-like protein, an NAD dependant epimerase and a dTDP-4-dehydrorhamnose 2–5 epimerase: they may be involved in rhamnose biosynthesis and glycosylation. The absence of these plasmid genes in B. hyodysenteriae isolates is predictive of reduced pathogenic potential.

Strains of the intestinal spirochaete Brachyspira pilosicoli attach to and aggregate erythrocytes

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species of mammals and birds, where it may induce colitis. Strains of the spirochaete have also been isolated from the bloodstream of immunocompromised human patients and have been seen in liver sections, and a similar systemic spread was recently observed in experimentally infected chickens. Some other spirochaete species that may be present in blood attach to and aggregate erythrocytes, and this is believed to contribute to disease severity. The aim of the current study was to determine whether B. pilosicoli strains have the capacity to attach to and aggregate erythrocytes. Initially, four strains of B. pilosicoli were incubated with erythrocytes from sheep, cows, pigs, dogs, humans, chickens and geese, and were observed by phase-contrast microscopy. Only strain WesB attached, and this was only with erythrocytes from chickens and geese. Subsequently, six other strains of B. pilosicoli were tested just with goose erythrocytes, and five attached to and caused aggregation of the erythrocytes. Scanning and transmission electron microscopy demonstrated that spirochaetes abutted and apparently firmly attached to the erythrocyte membranes. Aggregation of erythrocytes by B. pilosicoli may contribute to disease severity in species that develop a spirochaetaemia. The intestinal spirochaete Brachyspira pilosicoli has been isolated from the bloodstream of immunocompromised human patients, and spread to the liver has been reported in humans and in experimentally infected chickens. In this study, B. pilosicoli was shown to undergo attachment by one cell end to chicken and goose erythrocytes in vitro and to aggregate them. This activity has the potential to contribute to disease severity in avian and possibly other species that develop a spirochaetaemia and systemic spread. Avian erythrocytes may be useful for studying the mechanisms by which B. pilosicoli attaches to cells.

Dissemination of Clonal Groups of Brachyspira hyodysenteriae amongst Pig Farms in Spain, and Their Relationships to Isolates from Other Countries

BackgroundSwine dysentery (SD) is a widespread diarrhoeal disease of pigs caused by infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Understanding the dynamics of SD, and hence being able to develop more effective measures to counter its spread, depends on the ability to characterise B. hyodysenteriae variants and trace relationships of epidemic strains.Methodology/Principal FindingsA collection of 51 Spanish and 1 Portuguese B. hyodysenteriae isolates was examined using a multilocus sequence typing (MLST) scheme based on the sequences of seven conserved genomic loci. The isolates were allocated to 10 sequence types (STs) in three major groups of descent. Isolates in four of the STs were widely distributed in farms around Spain. One farm was infected with isolates from more than one ST. Sequence data obtained from PubMLST for 111 other B. hyodysenteriae strains from other countries then were included in the analysis. Two of the predominant STs that were found in Spain also were present in other European countries. The 73 STs were arranged in eleven clonal complexes (Cc) containing between 2 and 26 isolates. A population snapshot based on amino acid types (AATs) placed 75% of the isolates from 32 of the 48 AATs into one major cluster. The founder type AAT9 included 22 isolates from 10 STs that were recovered in Spain, Australia, Sweden, Germany, Belgium, the UK, Canada, and the USA.Conclusions/SignificanceThis MLST scheme provided sufficient resolution power to unambiguously characterise B. hyodysenteriae isolates, and can be recommended as a routine typing tool that rapidly enables comparisons of isolates. Using this method it was shown that some of the main genetic lineages of B. hyodysenteriae in Spain also occurred in other countries, providing further evidence for international transmission. Finally, analysis of AATs appeared useful for deducing putative ancestral relationships between strains.

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.

Evaluation of recombinant Brachyspira pilosicoli oligopeptide-binding proteins as vaccine candidates in a mouse model of intestinal spirochaetosis

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of humans, and various species of animals and birds, in which it may induce a mild colitis and diarrhoea. The aim of the current study was to evaluate the use of putative oligopeptide-binding proteins of B. pilosicoli as vaccine components. A partial genome sequence of B. pilosicoli porcine strain 95/1000 was subjected to bioinformatics analysis, and six genes predicted to encode oligopeptide-binding proteins were selected. Following a PCR-based distribution study of the genes across different strains of the spirochaete, they were amplified from B. pilosicoli human strain WesB and cloned in Escherichia coli. The recombinant histidine-tagged proteins were purified and subjected to in vitro and in vivo immunogenicity analysis. Recombinant products (P-1 and P-3) from two genes that were immunogenic and recognized by sera from pigs that had recovered from B. pilosicoli infections were tested in a mouse model of intestinal spirochaetosis. For each recombinant protein, groups of 12 C3H/HeJ mice were vaccinated subcutaneously with 100 microg protein emulsified in Freund's incomplete adjuvant, twice with a 2 week interval. Two weeks later the vaccinated and non-vaccinated control animals were challenged orally with B. pilosicoli strain WesB. Both proteins induced systemic and local colonic IgG antibody responses, and, following experimental infection, the cumulative number of colonization days was significantly (P<0.001) less in both groups of vaccinated mice compared to the control mice. There were significantly (P=0.012) fewer mice colonized in the group vaccinated with P-1 than in the non-vaccinated control group. The results suggest that oligopeptide-binding proteins may have potential for use as components of vaccines for B. pilosicoli.

Brachyspira intermedia strain diversity and relationships to the other indole-positive Brachyspira species

The aims of this study were to use multilocus sequence typing (MLST) to (i) investigate the population structure, diversity and molecular epidemiology of the weakly haemolytic anaerobic intestinal spirochaete Brachyspira intermedia, and (ii) determine the relationship of the species to the other two indole-positive but strongly haemolytic Brachyspira species— B. hyodysenteriae and “ B. suanatina”. Seventy-seven B. intermedia isolates from pigs and chickens were analysed, with the nucleotide sequences of seven conserved genomic loci examined for each. B. intermedia was genetically diverse, with the 77 isolates being divided into 71 sequence types (STs) and 64 amino acid types (AATs). Many distinct groups of B. intermedia isolates were identified, with some isolates being separated from others by large genetic distances. Although both pig and chicken isolates were found in most groups, suggesting that cross-species transmission of such isolates may occur, some isolates from pigs were located in small groups that did not include chicken isolates, and vice versa. Eight clonal complexes (Cc) of STs were identified by e-Burst analysis. The Ccs contained between 2 and 5 STs, and between 2 and 9 isolates. Five Ccs contained multiple isolates from the same farms, collected at the same time, indicating the existence of ongoing minor genetic change amongst isolates at the farm level. On the other hand, isolates with quite different STs also were found amongst multiple isolates collected from some farms. By comparison with the much more restricted diversity observed for 111 isolates of B. hyodysenteriae, and 4 isolates of “ B. suanatina”, it is difficult to justify including all weakly haemolytic indole-positive Brachyspira isolates in the single species B. intermedia.

Attraction of Brachyspira pilosicoli to mucin

The anaerobic intestinal spirochaete Brachyspira pilosicoli colonizes the large intestine of various species, including humans. In the colon this spirochaete can penetrate the overlying mucus layer, attach by one cell end to the underlying enterocytes, and initiate localized colitis and diarrhoea. The aim of this study was to investigate whether, as part of the colonization process, B. pilosicoli is attracted to mucin. Fifteen B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. No significant attraction was detected with 1 % mucin, but some strains started to enter a 2 % solution, and attraction then increased with increasing concentrations to peak at around 6-8 % mucin. A similar increase was seen with B. hyodysenteriae, although this activity peaked at 6 % mucin and then declined, suggesting that the two species have different affinities for mucin. These mucin concentrations were much higher than those used in previous experimental studies with Brachyspira species. The viscosities of the 6-8 % mucin solutions were around 7-12 mPa s, which were similar to the measured viscosities of the mucus layer overlying the epithelium in the caecum and colon of experimental pigs. The strains varied in their motility, as assessed by their ability to enter tubes containing chemotaxis buffer, but there was no significant relationship between this motility and the extent of their ability to enter the mucin solutions. Different strains also had different propensities to enter the mucin solutions, but there were no consistent differences according to the host species of origin. B. pilosicoli strain 95/1000 was attracted towards a solution of d-serine, suggesting that chemotaxis was involved in the attraction to mucin; however, 95/1000 was also attracted to viscous solutions of polyvinylpyrrolidone (PVP), in a manner mirroring the response to mucin, and hence suggesting the involvement of viscotaxis in the attraction to mucin. B. hyodysenteriae B204 showed a similar viscotaxis to PVP. Further studies are required to determine whether the in vitro interaction of a given strain with mucin is a useful indicator of its in vivo colonization ability, and hence could be used as a potential marker for virulence.

Isolation of the anaerobic intestinal spirochaete Brachyspira pilosicoli from long-term residents and Indonesian visitors to Perth, Western Australia

Brachyspira pilosicoli is an anaerobic spirochaete that colonizes the large intestine of humans and various species of animals and birds. The spirochaete is an important enteric pathogen of pigs and poultry, but its pathogenic potential in humans is less clear. In the current study, the occurrence of B. pilosicoli in faecal samples from 766 individuals in two different population groups in Perth, Western Australia, was investigated by selective anaerobic culture. Of 586 individuals who were long-term residents of Perth, including children, elderly patients in care and in hospital and individuals with gastrointestinal disease, only one was culture positive. This person had a history of diverticulitis. In comparison, faeces from 17 of 180 (9.4 %) Indonesians who were short- or medium-term visitors to Perth were positive for B. pilosicoli. The culture-positive individuals had been in the city for between 10 days and 4.5 years (median 5 months). Resampling of subsets of the Indonesians indicated that all negative people remained negative and that some positive individuals remained positive after 5 months. Two individuals had pairs of isolates recovered after 4 and 5 months that had the same PFGE types, whilst another individual had isolates with two different PFGE types that were identified 2 months apart. Individuals who were culture-positive were likely to have been either colonized in Indonesia before arriving in Perth or infected in Perth following contact with other culture-positive Indonesians with whom they socialized. Colonization with B. pilosicoli was not significantly associated with clinical signs at the time the individuals were tested, although faeces with wet-clay consistency were 1.5 times more likely (confidence interval 0.55-4.6) than normal faeces to contain B. pilosicoli.

A reverse vaccinology approach to swine dysentery vaccine development

Swine dysentery (SD) is a mucohaemorrhagic colitis of pigs resulting from infection of the large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Whole-cell bacterin vaccines are available to help control SD, but their performance has been inconsistent. This study aimed to use a reverse vaccinology approach to identify B. hyodysenteriae proteins for use as recombinant vaccine components. Nineteen open reading frames (ORFs) predicted to encode potential vaccine candidate molecules were identified from in silico analysis of partial genomic sequence data. The distribution of these ORFs among strains of B. hyodysenteriae was investigated by PCR, and widely distributed ORFs were cloned. The products were screened with a panel of immune pig sera, and from these a subset of conserved, immunogenic proteins was selected. Mice immunized intramuscularly with these recombinant proteins developed specific systemic antibody responses to them, and their sera agglutinated B. hyodysenteriae cells in vitro. In a pilot experiment, eight pigs were vaccinated twice intramuscularly with a combination of four of the proteins. The pigs developed antibodies to the proteins, and following experimental challenge only one developed SD compared to five of nine non-vaccinated control pigs. Although these differences in incidence were not significant, they indicated a trend towards protection using the recombinant proteins as immunogens. This study demonstrates that the reverse vaccinology approach has considerable potential for use in developing novel recombinant vaccines for SD.

An unexpectedly high prevalence of colonization with the intestinal spirochaete Brachyspira aalborgi amongst residents of the Indonesian island of Bali

PCR assays designed to amplify DNA from the anaerobic intestinal spirochaete Brachyspira aalborgi were conducted on DNA extracted from 938 faecal samples from 469 residents on the Indonesian island of Bali. The individuals tested were sampled twice in one year and were from four rural villages, one peri-urban centre and the capital city, Denpasar. Overall, an unexpectedly high prevalence of colonization (24.7%) was found, with prevalence rates at different locations varying from a low of 15.6% at one village to 41.5% in the peri-urban centre. Comparison of prevalence rates at the two sampling times suggested that, in many individuals, colonization was likely to be prolonged (>3 months) and/or that reinfection was occurring frequently in these people. Analysis of a questionnaire administered to the individuals who were sampled identified specific risk factors for colonization as location, co-colonization with the related intestinal spirochaete Brachyspira pilosicoli and use of drinking water obtained from wells rather than from taps. No specific associations with clinical symptoms were identified.

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 7.5 kb 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.

Penicillin resistance in the intestinal spirochaete Brachyspira pilosicoli associated with OXA-136 and OXA-137, two new variants of the class D β-lactamase OXA-63

Penicillin resistance mediated by beta-lactamase activity has been reported previously in the anaerobic intestinal spirochaete Brachyspira pilosicoli, and a novel class D beta-lactamase (OXA-63) hydrolysing oxacillin was described recently in a resistant human strain from France. In the current study, 18 B. pilosicoli strains from Australia and Papua New Guinea were tested for ampicillin and oxacillin susceptibility, and investigated for the presence of the class D beta-lactamase gene blaOXA-63 using PCR. PCR products were amplified from seven human and four porcine strains that were penicillin resistant, but not from seven penicillin-sensitive strains. Sequence analysis of the whole gene amplified from seven of the resistant strains from humans and pigs revealed only minor nucleotide differences among them, but there were significant differences compared with blaOXA-63. The predicted amino acid sequence of the enzyme from all seven strains had the same key structural motifs as the previously reported OXA-63, but two variants with 94-95% identity with OXA-63 were identified. OXA-136 had an additional amino acid and 12 other consistent amino acid substitutions compared with OXA-63. OXA-137 had the same differences compared with OXA-63 as OXA-136, but had an additional amino acid substitution at position 16. No structures consistent with integrons or transposons were found in the nucleotide sequences in the vicinity of blaOXA-136 in partially sequenced B. pilosicoli strain 95/1000, and the GC content (25.2 mol%) of the gene was similar to that of the whole genome. The gene encoding OXA-136 from B. pilosicoli strain Cof-10 conferred penicillin resistance on Escherichia coli. This study shows that penicillin resistance in human and porcine B. pilosicoli strains from Australia is associated with the production of two variants of OXA-63, and that susceptible strains lack the genes encoding OXA-63 or the variants.

Evaluation of recombinant Bhlp29.7 as an ELISA antigen for detecting pig herds with swine dysentery

Swine dysentery (SD) results from infection of the porcine large intestine with the anaerobic intestinal spirochaete Brachyspira hyodysenteriae. Diagnosis of SD traditionally has relied on detecting the spirochaete in the faeces of acutely affected pigs. To date simple and reliable serological assays that can be applied as a diagnostic tool at the herd level have not been available. In the current study a recombinant histidine tagged 29.7 kDa lipoprotein of B. hyodysenteriae (His 6-Bhlp29.7) was used as an ELISA plate-coating antigen. Sera ( n = 1121) from slaughter-aged pigs on 19 farms were tested in this ELISA. Following optimization of the ELISA conditions using hyperimmune control sera, a set of 464 sera from slaughter-aged pigs from five herds where SD did not occur was tested. From these results a suitable cut-off value for herd negativity was defined as the mean optical density reading plus three standard deviations. Testing of 337 pig sera from six farms with SD then showed that the sensitivity of the test at the herd level was 100%, with all six farms having one or more serum samples exceeding the cut-off value for negativity. Finally, 320 sera from eight herds suspected of having SD were examined. Four of these herds were shown to have pigs with titres consistent with SD. The true health status of the other four herds that were serologically negative could not be confirmed. In conclusion, when used on sets of 40 sera from slaughter-aged pigs the His 6-Bhlp29.7 ELISA as established proved to be a useful adjunct to the diagnosis of SD at the herd level.

The distribution of bmpB, a gene encoding a 29.7kDa lipoprotein with homology to MetQ, in Brachyspira hyodysenteriae and related species

The distribution of the bmpB gene encoding BmpB, a 29.7 kDa outer membrane lipoprotein of the intestinal spirochaete Brachyspira hyodysenteriae, was investigated. Using PCR, the gene was detected in all the 48 strains of B. hyodysenteriae examined and in Brachyspira innocens strain B256T, but not in 11 other strains of B. innocens nor in 42 strains of other Brachyspira spp. The gene was sequenced from B. innocens strain B256T and from 11 strains of B. hyodysenteriae. The B. hyodysenteriae genes shared 97.9-100% nucleotide sequence similarity and had 97.5-99.5% similarity with the gene of B. innocens strain B256T. Southern hybridisation indicated that bmpB was present on a 1.9 kb HindIII fragment of the B. hyodysenteriae genome and on a 3.1 kb fragment of the B. innocens B256T genome. The B. innocens lipoprotein did not react in Western blots with monoclonal antibody BJL/SH1 that reacts with the B. hyodysenteriae lipoprotein. The difference in binding with the monoclonal antibody may reside in the replacement of a serine residue with a tyrosine residue at base position 210 in the lipoprotein from B. innocens B256T. Comparison of the BmpB amino acid sequence with sequences in the SWISS-PROT protein database indicated that it has 33.9-39.9% similarity with the d-methionine binding proteins (MetQ) of a number of pathogenic bacterial species. The bmpB gene was confirmed to be the same as a gene of B. hyodysenteriae that was recently designated "blpA".

The wheat variety used in the diet of laying hens influences colonization with the intestinal spirochaete Brachyspira intermedia

This study investigated whether feeding different wheat varieties to laying hens could influence colonization with the intestinal spirochaete Brachyspira intermedia. Fifty ISA-Brown laying hens were divided into two groups. One group were fed a laying-hen diet formulated with wheat variety Westonia, and one were fed the diet incorporating variety Stilleto. Each group was divided into 15 hens experimentally infected with B. intermedia and 10 uninfected controls. The 30 infected hens were housed in individual cages in one room, and the controls were similarly housed in another room. Following administration of cultures of B. intermedia strain HB60 by crop-tube over 3 days, cloacal swabs were taken for spirochaete culture every 3 to 4 days. The water content of caecal faeces, and egg production and body weight were measured weekly. The hens were killed after 4 weeks, the caeca cultured for spirochaetes and the viscosity of the ileal contents measured. A total of 48/120 (40%) of the excreta samples from infected hens fed Westonia contained B. intermedia, compared with 21/120 (17.5%) for Stiletto (P=0.0002). The ileal viscosity of hens fed Westonia also was higher (P=0.048), but viscosity was not clearly related to the non-starch polysaccharide (NSP) content of the wheats. Westonia had a slightly higher total NSP content than Stiletto, but the ratio of soluble to insoluble NSP was lower. Infected hens developed wetter excreta, but neither infection nor diet altered egg production. In conclusion, the wheat variety can influence colonization with B. intermedia, apparently through diet-related alterations in the intestinal microenvironment.