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PubMed2004

A cross-sectional study to investigate the occurrence and distribution of intestinal spirochaetes (Brachyspira spp.) in three flocks of laying hens.

A cross-sectional study was conducted on a commercial egg-producing farm with a history of wet litter. A total of 600 fresh caecal faecal samples were obtained from under cages of laying hens in three sheds each containing flocks of approximately 5400 hens. Samples were cultured for intestinal spirochaetes, and growth on the primary isolation plate was observed under a phase contrast microscope and subjected to PCRs specific for the intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli. Spirochaete isolates obtained in pure culture were assessed for their ability to cause haemolysis on blood agar and to produce indole, and were typed using pulsed field gel electrophoresis (PFGE). A 1250 base pair portion of the 16S rRNA gene of three B. intermedia and five unidentified isolates was sequenced, and the sequences compared with those of other Brachyspira species. Overall, 121 (20.2%) of the faecal samples contained spirochaetes as determined by growth on the plate and microscopy. Using PCR on the primary growth from these positive samples, 43 (7.2% overall) were shown to contain B. intermedia, 8 (1.3%) to contain B. pilosicoli, and 70 (11.7%) were PCR negative. Only 24 isolates of B. intermedia and five isolates of unknown species were obtained in pure culture. Comparative analysis of the 16S rRNA gene sequence identified the non-B. intermedia isolates as belonging to the proposed species "Brachyspira pulli". PFGE analysis of the B. intermedia strains identified them as having four major banding patterns. Individual patterns were found in hens from different flocks, suggesting cross-transmission of strains between flocks. No environmental sources of infection were identified. The youngest flock had a significantly lower level of colonisation with B. intermedia than the flock of intermediate age (P = 0.004), suggesting that following initial infection of individual young hens on this farm there was amplification and transmission of infection amongst members of the flock.

Age Factors↗
PubMed2004

Rapid isolation of Brachyspira hyodysenteriae and Brachyspira pilosicoli from pigs.

The aim of this study was to compare and evaluate the time required to isolate Brachyspira hyodysenteriae and Brachyspira pilosicoli from porcine faeces. This was done using previously described selective media (spectinomycin) S400, (colistin, vancomycin and spectinomycin) CVS and (spectinomycin, vancomycin, colistin, spiramycin and rifampin with swine faecal extract) BJ, compared with the method based on blood agar modified medium, with spectinomycin and rifampin (BAM-SR), including a pre-treatment step. Fourteen spirochaetal strains were obtained in pure cultures after 5 days (48 h in BAM-SR primary plate and three passages every 24 h in brain heart infusion (BHI) without antibiotics) pre-treating simulated samples in brain heart infusion broth with spectinomycin (400 microg/ml) and rifampin (15 microg/ml), before streaking on the selective BAM-SR medium. Spirochaetes from samples in S400, CVS and BJ, with and without pre-treatment, were obtained in pure cultures only after repeatedly transferring on plates of the same selective medium requiring 15-18 days according to the strain. BAM-SR used after the pre-treatment step showed a detection limit ranging from 3.5 x 10(2) to 6.7 x 10(7) cells/g faeces and was the only method able to support the growth of spirochaetes after 48 h.

Animals↗
PubMed2005

The use of multilocus enzyme electrophoresis to characterise intestinal spirochaetes (Brachyspira spp.) colonising hens in commercial flocks.

Multilocus enzyme electrophoresis (MLEE) was used to identify, examine genetic relationships and look at disease associations of a collection of 53 intestinal spirochaete isolates previously recovered from the faeces of adult hens on 14 farms in Qld, Australia. The MLEE results were compared with those previously obtained using species-specific PCR amplifications. The isolates were divided into five Brachyspira species groups by MLEE: Brachyspira murdochii (n=17), B. intermedia (n=15), B. pilosicoli (n=14), B. innocens (n=2) and "B. pulli" (n=1). Three new MLEE groups each containing single isolates also were identified. The results of the PCR assay for B. pilosicoli were concordant with the MLEE results, but the 23S rDNA-based PCR for B. intermedia had failed to detect 8 of the 15 isolates. The B. innocens/B. murdochii nox-based PCR had correctly identified all the isolates of B. murdochii, but did not identify either of the two B. innocens isolates. Using MLEE, isolates from two farms (14%) were identified as B. murdochii, whilst the pathogenic species B. intermedia and B. pilosicoli were present in hens from eight (57%) and five (36%) farms, respectively, and were identified together in four (29%) farms. All seven of the farms with production problems or wet litter were colonised with B. intermedia and/or B. pilosicoli. Six farms had multiple spirochaete isolates available for examination. Two broiler breeder farms both had five isolates of B. pilosicoli that shared the same MLEE electrophoretic type (ET), whilst one laying hen farm had three isolates of B. intermedia that all belonged to the same ET. Hence on each of these farms a predominant strain of a pathogenic species was present. On the other farms isolates of the same species were more diverse and belonged to different ETs. These results show that the epidemiology of intestinal spirochaetal infections in broiler breeder and laying hen flocks can vary considerably between farms, although the reasons for these differences were not established.

Animals↗
PubMed2005

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.

Animals↗
PubMed2006

Development of a two-step nested duplex PCR assay for the rapid detection of Brachyspira pilosicoli and Brachyspira intermedia in chicken faeces.

Avian intestinal spirochaetosis (AIS) is an infection of the caeca and/or colo-rectum of laying and meat breeder hens caused by anaerobic intestinal spirochaetes of the genus Brachyspira. AIS can result in a variety of symptoms, including delayed and/or reduced egg production, and increased faecal water content. The two most commonly reported Brachyspira species involved in AIS are Brachyspira pilosicoli and Brachyspira intermedia, and their detection and identification can be difficult and time consuming. In the current study a two-step nested duplex PCR (2S-N-D-PCR) was developed for the detection of these two species, using DNA extracted from washed chicken faeces. In the first step, a duplex PCR (D-PCR) amplifying Brachyspira genus-specific portions of the 16S rRNA and NADH oxidase (nox) genes was undertaken on the washed faeces. In the second step, a nested D-PCR was used that amplified species-specific portions of the 16S rRNA gene of B. pilosicoli and the nox gene of B. intermedia from the amplicons produced in the first step. The 2S-N-D-PCR was rapid and specific, and could be used to detect approximately 10(3) cells of each spirochaete species per gram of washed faeces. When tested on 882 chicken faecal samples from infected flocks, it detected 4-5% more positive faecal samples than did the standard method of selective anaerobic culture followed by individual species-specific PCR assays conducted on the growth on the primary plate. The application of this new technique should improve diagnostic capacity, and facilitate further studies on AIS.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

The effect of fermentable carbohydrates on experimental swine dysentery and whip worm infections in pigs.

An experiment was conducted to study the effect of diets with contrasting fermentability in the large intestine on experimental infections with Brachyspira hyodysenteriae, the causative agent of swine dysentery, and the whip worm, Trichuris suis, in pigs. Two diets with organically grown ingredients were composed. Both diets were based on triticale and barley and supplemented with either rape seed cake (Diet 1) or dried chicory root and sweet lupins (Diet 2). The study had a three-factorial design, with eight groups of pigs receiving Diet 1 or Diet 2, +/-B. hyodysenteriae, and +/-T. suis. Pigs fed Diet 2 and challenged with B. hyodysenteriae did not develop swine dysentery and B. hyodysenteriae was not demonstrated in any of the pigs during the study. In contrast, 94% of the B. hyodysenteriae challenged pigs fed Diet 1 showed clinical symptoms of swine dysentery and all the pigs were shedding B. hyodysenteriae in faeces at some points in time during the experiment. The number of T. suis was lower in pigs fed Diet 2 compared to pigs fed Diet 1, but the differences were not significant. Pigs on Diet 1 and challenged with both pathogens showed clinical symptoms of SD for a longer period than pigs inoculated with B. hyodysenteriae only. The study showed that diets supplemented with highly fermentable carbohydrates from dried chicory roots and sweet lupins can protect pigs against developing swine dysentery, but do not have any significant influence on T. suis.

Animal Feed↗
PubMed2003

Differential requirements for proliferation of CD4+ and gammadelta+ T cells to spirochetal antigens.

Alphabeta+ and gammadelta+ T cells have different mechanisms of epitope recognition and are stimulated by antigens of different chemical nature. An immunization model with antigens from the spirochete Brachyspira hyodysenteriae was used to examine the requirements for proliferation of circulating porcine CD4+ and gammadelta+ T cells in mixed lymphocyte cultures. CD4+ T cells only responded to stimulation with B. hyodysenteriae antigens, whereas gammadelta+ T cells proliferated when cultures were stimulated with either spirochetal antigens or interleukin-2 (IL-2). T cells that had proliferated expressed high levels of IL-2-receptor-alpha (IL-2Ralpha). Furthermore, neutralization of IL-2 at the beginning of the culture period was more efficient in blocking gammadelta+ than CD4+ T cell proliferation. Immunization induced interferon-gamma (IFN-gamma) production by CD4+ T cells, whereas only a small fraction of the antigen-stimulated gammadelta+ T cells produced this cytokine. Our results indicate that, under the same environmental conditions, CD4+ T cell functions are more tightly regulated when compared to gammadelta+ T cells. We conclude that these differences are due, in part, to the enhanced gammadelta+ T cell responsiveness to IL-2.

Antigens, Bacterial↗
PubMed1985

Intestinal spirochetosis of the vermiform appendix.

A series of 681 surgically removed appendices were examined for spirochetes. Five hundred seventy-four appendices were removed because of suspected acute appendicitis; the rest were removed per occasionem. One hundred six of the former were histologically normal, whereas 421 showed acute appendicitis. The remaining 47 specimens showed a variety of other pathological conditions, for example, tumors and diverticula. Spirochetes were found in 13 (12.3%) of the appendices removed from patients clinically suspected to suffer from acute appendicitis, but whose appendices were otherwise histologically normal (pseudoappendicitis). Only 3 patients (0.7%) with histologically confirmed acute appendicitis (p less than 0.05) did show spirochetes in their appendices. Of the 107 patients who had their appendices removed per occasionem, 2 patients (1.9%) had spirochetosis (p less than 0.05). The ultrastructure of the spirochetes obtained from appendices with spirochetosis was studied by means of negative staining and ultrathin sectioning. The morphology of these spirochetes was very similar to that of Brachyspira aalborgi, a spirochete recently isolated from rectal biopsy specimens obtained from patients with colorectal spirochetosis.

Appendectomy↗
PubMed1985

Isolation of spirochetes from the skin of patients with erythema chronicum migrans in Austria.

Spirochetal organisms were isolated from biopsies (skin punches) of the erythematous anular skin lesion of three patients with erythema chronicum migrans. The organisms were cultivated in modified Kelly's medium and - after an incubation period of 2 weeks - discovered by dark field microscopy (X 200). Compared with the Lyme disease agent Borrelia burgdorferi the organisms did not differ in shape, motility and reaction with polyclonal antiserum. Differences to B. burgdorferi were found in (i) very slow growth of the isolated organisms in modified Kelly's medium and in (ii) non reactivity with monoclonal antibodies against the outer membrane protein of B. burgdorferi. It is concluded that the Austrian isolates are closely related to but not identical with the Lyme disease agent.

Adult↗
PubMed1987

Intestinal spirochaetosis. Light and electron microscopic study.

24 cases of colon spirochaetosis have been studied by light and electron microscopy. This condition, although rarely diagnosed because of the absence of special clinical symptoms, seems to be a rather frequent histologic finding with marked variation in its geographical incidence. In our material the incidence of spirochaetosis was 16.5%, almost double than in Great Britain.

Adult↗
PubMed2002

Dietary enzyme and zinc bacitracin reduce colonisation of layer hens by the intestinal spirochaete Brachyspira intermedia.

Brachyspira intermedia strain HB60 was used to experimentally infect 40 individually caged 22-week-old laying hens. Another 10 control birds were sham-inoculated with sterile broth. All chickens received an experimental layer diet based on wheat. The infected birds were randomly divided into four groups of 10, with the diet for each group containing either 50 ppm zinc bacitracin (ZnB), 100 ppm ZnB, 256 ppm of dietary enzyme (Avizyme), 1302), or no additive. Birds were kept for 6 weeks after infection, and faecal excretion of B. intermedia, faecal water content, egg numbers, egg weights and body weights were recorded weekly. Control birds remained uninfected throughout the experiment. B. intermedia was isolated significantly less frequently from the groups of experimentally infected birds receiving ZnB at 50 ppm or enzyme than from those receiving 100 ppm ZnB or no treatment. Infected birds had a transient increase in faecal water content in the week following challenge, but no other significant production differences were detected amongst the five groups of birds in subsequent weeks. It was not established how the ZnB at 50 ppm and the dietary enzyme reduced the ability of the spirochaete to colonise, but it may have been by bringing about changes in the intestinal microflora and/or the intestinal microenvironment.

Animal Feed↗
PubMed1998

Detection of Lawsonia intracellularis, Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes, Salmonella enterica, and haemolytic Escherichia coli from swine herds with and without diarrhoea among growing pigs.

A polymerase chain reaction (PCR) was optimized to detect Lawsonia intracellularis in faeces from naturally infected pigs. By combining a boiling procedure to extract DNA and a nested PCR procedure, a detection limit at 2 x 10(2) bacterial cells per gram of faeces was achieved. The optimized PCR was used together with conventional culture techniques to detect Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes (WBHIS), Salmonella enterica, and haemolytic Escherichia coli, in a case control study to examine selected risk factors for the development of diarrhoea in growing pigs. Herds with diarrhoea were selected as cases and randomly chosen herds without diarrhoea were chosen as controls. Infection with L. intracellularis significantly enhanced the chance of diarrhoea. S. hyodysenteriae, WBHIS group IV (Serpulina pilosicoli), and S. enterica were isolated only from case herds which indicate that these species may influence the development of diarrhoea. In addition, herd-type had a significant impact, that is specific pathogen-free herds showed an odds ratio at 0.2 relative to conventional herds for the development of diarrhoea.

Animals↗
PubMed2000

Phylogenetic evidence for novel and genetically different intestinal spirochetes resembling Brachyspira aalborgi in the mucosa of the human colon as revealed by 16S rDNA analysis.

Intestinal spirochetes (Brachyspira spp.) are causative agents of intestinal disorders in animals and humans. Phylogenetic analysis of cloned 16S rRNA genes from biopsies of the intestinal mucosa of the colon from two Swedish 60-years old adults without clinical symptoms revealed the presence of intestinal spirochetes. Seventeen clones from two individuals and 11 reference strains were analyzed and the intestinal spirochetes could be divided into two lineages, the Brachyspira aalborgi and the Brachyspira hyodysenteriae lineages. All of the clones grouped in the B. aalborgi lineage. Moreover, the B. aalborgi lineage could be divided into three distinct phylogenetic clusters as confirmed by bootstrap and signature nucleotide analysis. The first cluster comprised 6 clones and the type strain B. aalborgi NCTC 11492T. The cluster 1 showed a 16S rRNA gene similarity of 99.4-99.9%. This cluster also harbored the only other strain of B. aalborgi isolated so far, namely strain W1, which was subjected to phylogenetic analysis in this work. The second cluster harbored 9 clones with a 98.7 to 99.5% range of 16S rDNA similarity to the B. aalborgi cluster 1. Two clones branched distinct and early of the B. aalborgi line forming the third cluster and was found to be 98.7% similar to cluster 1 and 98.3-99.1% to cluster 2. Interestingly, this shows that considerable variation of intestinal spirochetes can be found as constituents of the colonic microbiota in humans, genetically resembling B. aalborgi. The presented data aid significantly to the diagnostic and taxonomic work on these organisms.

Cell Lineage↗
PubMed2003

Rapid detection and identification of Brachyspira aalborgi from rectal biopsies and faeces of a patient.

This study reports for the first time the detection of Brachyspira aalborgi in faeces and rectal biopsies of a female suffering for 3-4 months of abdominal pain with long-standing mucosal diarrhoea, rectal bleeding and suspected carcinoma of the rectum. After pre-treatment of samples (faeces and biopsies) with a liquid medium (trypticase soy broth-TSB) containing foetal calf serum (FCS, 10%) and spectinomycin and rifampicin (TSB-SR) the first detection of B. aalborgi isolate HBS1 was observed after 48 h in the primary plates of selective blood agar modified medium (BAM) containing spectinomycin and rifampicin (BAM-SR), where growth zones were signalled by a small weakly beta-haemolytic halo. Attempts to subculture spirochaetes in agar media failed. The new HBS1 isolate was only propagated in TSB broth and at electron microscopy it showed 4 endoflagella inserted at each tapered end. The phenotypic characterization of HBS1 demonstrated absence of hippurate hydrolysis, indole production, alpha-galactosidase, alpha- and beta-glucosidase activities in accordance with the B. aalborgi type strain. Rapid identification of B. aalborgi isolate HBS1 was performed directly from faeces and rectal biopsies and subsequently from pure cultures by a genetic method based on 16S DNA restriction fragment length polymorphism (RFLP)-polymerase chain reaction (PCR). The sequence of 16S DNA amplicon of the isolate HBS1 was found 99.2% corresponding to that of the B. aalborgi type strain. Our results encourage further investigations for the development of a suitable selective agar medium for the isolating and cultivating B. aalborgi from human specimens.

Biopsy↗
PubMed1990

Morphological heterogeneity among spirochetes isolated from cases of swine dysentery.

Pathogenic and non-pathogenic spirochetes isolated from the intestines of pigs were examined by electron microscopy using the negative staining and ultrathin sectioning techniques. Morphological differences were observed among cells of different strains. The cells differed in length as well as in width and in the number of flagella inserted at each end. In addition, the cells from different strains also varied in their resistance to the action of the detergents, Teepol and sodium deoxycholate. Three of the strains studied contained weakly haemolytic spirochetes, two of which differed markedly in their morphology from the cells of the other strains. These spirochetes had fewer flagella inserted at each end than those from other isolates and showed a distinct lattice-like substructure covering the ends of the cells. The spirochetes examined were found to be morphologically more similar to those of the genus Borrelia than to those of the genus Treponema but were clearly different from the cells of both of these genera. The taxonomic implications of the observations are discussed in brief.

Animals↗