In vitro attenuation of a virulent swine isolate of Brachyspira hampsonii.

Brachyspira hampsonii causes dysentery-like disease in infected pigs. Serial passage of a virulent swine isolate (P13) one-hundred times in laboratory culture medium was conducted to produce an attenuated strain, and to identify genomic determinants of virulence through comparison of genome sequences of the original and passaged strains. The resulting strain, P113, did not differ from P13 in terms of diagnostic biochemical characteristics but had an enhanced growth rate in culture, indicating laboratory adaptation. Whole genome sequencing of P113 revealed several single-nucleotide changes including a T to C transition that results in an R to G amino acid change in a putative mannose-1-phosphate guanylytransferase that is implicated in production of lipo-oligosaccharide. P113 was partially attenuated in a mouse model of infection, indicated by significantly fewer observations of abnormal feces in mice infected with P113 relative to P13. No differences were detected in bacterial shedding in feces, demonstrating that the ability of the organism to colonize mice was not affected. Passage through a mouse did not further alter the virulence of P113. Results of this study provide insight into genomic determinants of virulence in B. hampsonii and a live attenuated vaccine candidate.

Metabonomics-based analysis of Brachyspira pilosicoli's response to tiamulin reveals metabolic activity despite significant growth inhibition.

Pathogenic anaerobes Brachyspira spp. are responsible for an increasing number of Intestinal Spirochaetosis (IS) cases in livestock against which few approved treatments are available. Tiamulin is used to treat swine dysentery caused by Brachyspira spp. and recently has been used to handle avian intestinal spirochaetosis (AIS). The therapeutic dose used in chickens requires further evaluation since cases of bacterial resistance to tiamulin have been reported. In this study, we evaluated the impact of tiamulin at varying concentrations on the metabolism of B. pilosicoli using a 1H-NMR-based metabonomics approach allowing the capture of the overall bacterial metabolic response to antibiotic treatment. Based on growth curve studies, tiamulin impacted bacterial growth even at very low concentration (0.008 µg/mL) although its metabolic activity was barely affected 72 h post exposure to antibiotic treatment. Only the highest dose of tiamulin tested (0.250 µg/mL) caused a major metabolic shift. Results showed that below this concentration, bacteria could maintain a normal metabolic trajectory despite significant growth inhibition by the antibiotic, which may contribute to disease reemergence post antibiotic treatment. Indeed, we confirmed that B. pilosicoli remained viable even after exposition to the highest antibiotic dose. This paper stresses the need to ensure new evaluation of bacterial viability post bacteriostatic exposure such as tiamulin to guarantee treatment efficacy and decrease antibiotic resistance development.

Brachyspira and its role in avian intestinal spirochaetosis.

The fastidious, anaerobic spirochaete Brachyspira is capable of causing enteric disease in avian, porcine and human hosts, amongst others, with a potential for zoonotic transmission. Avian intestinal spirochaetosis (AIS), the resulting disease from colonisation of the caeca and colon of poultry by Brachyspira leads to production losses, with an estimated annual cost of circa £ 18 million to the commercial layer industry in the United Kingdom. Of seven known and several proposed species of Brachyspira, three are currently considered pathogenic to poultry; B. alvinipulli, B. intermedia and B. pilosicoli. Currently, AIS is primarily prevented by strict biosecurity controls and is treated using antimicrobials, including tiamulin. Other treatment strategies have been explored, including vaccination and probiotics, but such developments have been hindered by a limited understanding of the pathobiology of Brachyspira. A lack of knowledge of the metabolic capabilities and little genomic information for Brachyspira has resulted in a limited understanding of the pathobiology. In addition to an emergence of antibiotic resistance amongst Brachyspira, bans on the prophylactic use of antimicrobials in livestock are driving an urgent requirement for alternative treatment strategies for Brachyspira-related diseases, such as AIS. Advances in the molecular biology and genomics of Brachyspira heralds the potential for the development of tools for genetic manipulation to gain an improved understanding of the pathogenesis of Brachyspira.

Occurrence of viable Brachyspira spp. on carcasses of spent laying hens from supermarkets.

Brachyspira spp. are frequent inhabitants of the chicken's intestine and some have been associated with enteric disease in humans. We studied contamination with Brachyspira spp. of carcasses of spent laying hens as a possible source of infections for humans and animals that may eat this meat. Eleven batches of hen carcasses, for a total of 110 carcasses, were bought in Belgian supermarkets during 2009-2010. Carcass rinse samples were examined for the presence of Brachyspira. Brachyspira spp. were cultured from some carcass in all batches. Besides presumably non-pathogenic species such as Brachyspira murdochii and Brachyspira innocens, the poultry pathogen Brachyspira intermedia and the poultry and suspected human pathogen Brachyspira pilosicoli were identified in 7/11 and 1/11 carcass batches, respectively, at high numbers, as shown using quantitative polymerase chain reactions. Multilocus sequence typing (MLST) demonstrated the presence of 2 and 13 MLST types of B. pilosicoli and B. intermedia, respectively, with all strains belonging to novel MLST types. The findings show that carcasses of spent laying hens are commonly contaminated with high numbers of Brachyspira spp., including the suspected zoonotic agent B. pilosicoli.

Analysis of bacterial load and prevalence of mixed infections with Lawsonia intracellularis, Brachyspira hyodysenteriae and/or Brachyspira pilosicoli in German pigs with diarrhoea.

Lawsonia (L.) intracellularis, Brachyspira (B.) hyodysenteriae and B. pilosicoli are important pathogens in domestic pig production world-wide, responsible for porcine intestinal adenomatosis, swine dysentery, and porcine intestinal spirochetosis, respectively. Conventional PCR is the major diagnostic tool in the detection of the three pathogens, but the sole detection of bacterial DNA might lead to misinterpretations of results with respect to their clinical relevance, especially with mixed infections. Thus, the present study targeted the detection and quantification of the three pathogens in samples from herds with a case history of diarrhoea. Herds and samples were selected by the practitioners on a voluntary basis. Results were based on 1176 individual samples from 95 herds from Southern Germany. The pathogens were detected simultaneously by multiplex real-time PCR. The overall prevalence for L. intracellularis, B. hyodysenteriae and B. pilosicoli was 12.6%, 8.4% and 3.2% in faecal samples and 48.4%, 24.2% and 31.6% in herds, respectively. Sixty one percent, 82.6%, and 73.4% of herds positive for L. intracellularis, B. hyodysenteriae, and B. pilosicoli, respectively, had mixed infections. Median log values of DNA equivalents/g of faeces for L. intracellularis, B. hyodysenteriae and B. pilosicoli were 3.3, 5.9 and 3.2, with maxima of 8.3, 8.0 and 6.3, respectively. Within herd prevalence of B. hyodysenteriae and B. pilosicoli as well as the load of B. hyodysenteriae were significantly associated with the severity of diarrhoea.

Lactobacilli antagonize the growth, motility, and adherence of Brachyspira pilosicoli: a potential intervention against avian intestinal spirochetosis.

Avian intestinal spirochetosis (AIS) results from the colonization of the ceca and colorectum of poultry by pathogenic Brachyspira species. The number of cases of AIS has increased since the 2006 European Union ban on the use of antibiotic growth promoters, which, together with emerging antimicrobial resistance in Brachyspira, has driven renewed interest in alternative intervention strategies. Probiotics have been reported as protecting livestock against infection with common enteric pathogens, and here we investigate which aspects of the biology of Brachyspira they antagonize in order to identify possible interventions against AIS. The cell-free supernatants (CFS) of two Lactobacillus strains, Lactobacillus reuteri LM1 and Lactobacillus salivarius LM2, suppressed the growth of Brachyspira pilosicoli B2904 in a pH-dependent manner. In in vitro adherence and invasion assays with HT29-16E three-dimensional (3D) cells and in a novel avian cecal in vitro organ culture (IVOC) model, the adherence and invasion of B. pilosicoli in epithelial cells were reduced significantly by the presence of lactobacilli (P < 0.001). In addition, live and heat-inactivated lactobacilli inhibited the motility of B. pilosicoli, and electron microscopic observations indicated that contact between the lactobacilli and Brachyspira was crucial in inhibiting both adherence and motility. These data suggest that motility is essential for B. pilosicoli to adhere to and invade the gut epithelium and that any interference of motility may be a useful tool for the development of control strategies.

Exposure to norepinephrine enhances Brachyspira pilosicoli growth, attraction to mucin and attachment to Caco-2 cells.

Brachyspira pilosicoli is an anaerobic intestinal spirochaete that colonizes the large intestine of a variety of species of birds and mammals, including human beings. Colonization may result in a mild colitis and diarrhoea in a condition known as 'intestinal spirochaetosis'. The catecholamine norepinephrine (NE), which is known to influence the behaviour of many bacterial species, may be present in the colon. The purpose of the current study was to determine whether exposure of B. pilosicoli to NE would influence its in vitro behaviour in assays that may reflect in vivo colonization potential. B. pilosicoli strain 95/1000 was used in all the assays. Addition of NE at a concentration of 0.05 mM to B. pilosicoli growing in anaerobic broth significantly increased spirochaete numbers after 4 days incubation. The effect of higher concentrations of NE was not significant. Exposure to 0.05 mM NE, but not to higher concentrations, also resulted in significantly more spirochaete cells entering capillary tubes containing 4 % porcine gastric mucin than occurred with untreated cultures. When NE was added to chemotaxis buffer in capillary tubes, significantly more spirochaetes were attracted to the buffer containing NE at 0.1, 0.5 and 1.0 mM than to buffer containing 0.05 mM NE, or when no NE was added. Exposure of B. pilosicoli cultures to 0.05 mM NE prior to incubation with Caco-2 monolayers resulted in more attachment to the monolayer than occurred with non-exposed cultures. These results show that at higher concentrations, NE acts as a chemoattractant for B. pilosicoli, and at 0.05 mM it increases the spirochaete's growth rate, attraction to mucin and rate of attachment to cultured enterocytes. These activities are likely to enhance the ability of B. pilosicoli to colonize, and may be induced by conditions that increase NE concentrations in the intestinal tract, such as the stresses associated with crowding.

Identification of Brachyspira hyodysenteriae and other pathogenic Brachyspira species in chickens from laying flocks with diarrhea or reduced production or both.

Cecal samples from laying chickens from 25 farms with a history of decreased egg production, diarrhea, and/or increased feed conversion ratios were examined for anaerobic intestinal spirochetes of the genus Brachyspira. Seventy-three samples positive in an immunofluorescence assay for Brachyspira species were further examined using selective anaerobic culture, followed by phenotypic analysis, species-specific PCRs (for Brachyspira hyodysenteriae, B. intermedia, and B. pilosicoli), and a Brachyspira genus-specific PCR with sequencing of the partial 16S rRNA gene products. Brachyspira cultures were obtained from all samples. Less than half of the isolates could be identified to the species level on the basis of their biochemical phenotypes, while all but four isolates (5.2%) were speciated by using PCR and sequencing of DNA extracted from the bacteria. Different Brachyspira spp. were found within a single flock and also in cultures from single chickens, emphasizing the need to obtain multiple samples when investigating outbreaks of avian intestinal spirochetosis. The most commonly detected spirochetes were the pathogenic species B. intermedia and B. pilosicoli. The presumed nonpathogenic species B. innocens, B. murdochii, and the proposed "B. pulli" also were identified. Pathogenic B. alvinipulli was present in two flocks, and this is the first confirmed report of B. alvinipulli in chickens outside the United States. Brachyspira hyodysenteriae, the agent of swine dysentery, also was identified in samples from three flocks. This is the first confirmed report of natural infection of chickens with B. hyodysenteriae. Experimental infection studies are required to assess the pathogenic potential of these B. hyodysenteriae isolates.

Asian origin and rapid global spread of the destructive dry rot fungus Serpula lacrymans.

The dry rot fungus Serpula lacrymans (Basidiomycota) is the most damaging destroyer of wood construction materials in temperate regions. While being a widespread aggressive indoor biodeterioration agent, it is only found in a few natural environments. The geographical source of spread and colonization by this fungus in human environments is thus somewhat of an enigma. Employing genetic markers (amplified fragment length polymorphisms, DNA sequences and microsatellites) on a worldwide sample of specimens, we show that the dry rot fungus is divided into two main lineages; one nonaggressive residing naturally in North America and Asia (var. shastensis), and another aggressive lineage including specimens from all continents, both from natural environments and buildings (var. lacrymans). Our genetic analyses indicate that the two lineages represent well-differentiated cryptic species. Genetic analyses pinpoint mainland Asia as the origin of the aggressive form var. lacrymans. A few aggressive genotypes have migrated worldwide from Asia to Europe, North and South America and Oceania followed by local population expansions. The very low genetic variation in the founder populations indicate that they have established through recent founder events, for example by infected wood materials transported over land or sea. A separate colonization has happened from mainland Asia to Japan. Our data also indicate that independent immigration events have happened to Oceania from different continents followed by admixture.

Colonic spirochetosis in animals and humans.

Colonic spirochetosis is a disease caused by the gram-negative bacteria Brachyspira aalborgi and Brachyspira pilosicoli. B. pilosicoli induces disease in both humans and animals, whereas B. aalborgi affects only humans and higher primates. Symptoms in humans include diarrhea, rectal bleeding, and abdominal cramps. Colonic spirochetosis is common in third world countries; however, in developed countries, the disease is observed mainly in homosexual males. Terminally ill patients infected with Brachyspira are particularly at risk for developing spirochetemia. Diarrhea, poor growth performance, and decreased feed-to-gain efficiency is seen in pigs with colonic spirochetosis. The disease in chickens is characterized by delayed and/or reduced egg production, diarrhea, poor feed conversion, and retarded growth. Thus, colonic spirochetosis can represent a serious economic loss in the swine and poultry industries. The organisms are transmitted by the fecal-oral route, and several studies have demonstrated that human, primate, pig, dog, or bird strains of B. pilosicoli can be transmitted to pigs, chickens, and mice. B. pilosicoli may be a zoonotic pathogen, and although it has not been demonstrated, there is a possibility that both B. pilosicoli and B. aalborgi can be transferred to humans via contact with the feces of infected animals, meat from infected animals, or food contaminated by food handlers. Neither B. pilosicoli nor B. aalborgi has been well characterized in terms of basic cellular functions, pathogenicity, or genetics. Studies are needed to more thoroughly understand these Brachyspira species and their disease mechanisms.

Brachyspira pilosicoli colonization in experimentally infected mice can be facilitated by dietary manipulation.

The purpose of this study was to determine whether defined dietary manipulations would enhance colonization of mice experimentally challenged with the intestinal spirochaete Brachyspira pilosicoli. Weanling C3H/HeJ mice (n = 48) were fed either a standard balanced mouse diet or a diet supplemented with 50 p.p.m. zinc bacitracin (ZnB), with 50 % (w/w) lactose or with both supplements. Eight mice from each group were challenged orally with a human strain of B. pilosicoli (WesB), whilst four in each group acted as uninfected controls to evaluate the effects of the diets alone. The mice were kept for 40 days following challenge and faeces were collected every 3-4 days and subjected to culture and PCR for B. pilosicoli. Feeding ZnB reduced total volatile fatty acid production in the caecum. Feeding lactose alone doubled the weight of the caecum and its contents compared with control mice, and resulted in a relatively greater production of acetate, but a reduction in propionate and isovalerate production. These effects were negated by the addition of ZnB with the lactose. None of the mice fed the standard diet or receiving ZnB alone became colonized following experimental challenge. One of the mice receiving lactose was culture and PCR positive for B. pilosicoli on one sampling 1 week after inoculation, and one was positive on three samplings taken 20, 25 and 29 days after inoculation. All mice receiving both lactose and ZnB became colonized and remained so, with all samples being positive over the last seven samplings. The colonization was not associated with an end-on attachment of the spirochaete to the epithelial cells of the caecum, but colonized mice developed a specific humoral antibody response to the spirochaete.

Survival of intestinal spirochaete strains from chickens in the presence of disinfectants and in faeces held at different temperatures.

This study aimed to evaluate the efficacy of some commonly used disinfectants in inactivating the pathogenic avian intestinal spirochaetes Brachyspira intermedia and Brachyspira pilosicoli, and to examine spirochaete survival in chicken caecal faeces held at 4 degrees C, 25 degrees C or 37 degrees C. Six disinfectants were evaluated at their recommended working concentrations: alkaline salts, quaternary ammonium, iodine as an iodophor, chlorine from a chlorine-release agent, glutaraldehyde and hydrogen peroxide. All but alkaline salts inactivated two different concentrations of both spirochaete species in less than 1 min in the presence of organic matter. Both spirochaete species at three different cell concentrations survived in caecal faeces at 37 degrees C for between 2 and 17 h. B. intermedia tended to survive for longer than B. pilosicoli, but the maximum survival time for both species at 4 degrees C was only 72 to 84 h. Hence, avian intestinal spirochaetes are rapidly inactivated by several common disinfectants, and their survival time in chicken caecal faeces is much less than has been reported for porcine intestinal spirochaetes in porcine faeces. It should be relatively easy to break the cycle of infection between batches of laying birds by resting sheds for a few days, and by using disinfectants on any residual faecal matter.

Antimicrobial susceptibility testing of porcine Brachyspira (Serpulina) species isolates.

No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 micro g/ml), tylosin (>256 micro g/ml), erythromycin (>256 micro g/ml), clindamycin (>4 micro g/ml), virginiamycin (4 micro g/ml), and carbadox (0.06 micro g/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.

Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochetosis.

Research in the past decade has led to the recognition of Brachyspira (formerly Serpulina) pilosicoli as the primary etiologic agent of colonic spirochetosis (CS), an emerging cause of colitis in humans and animals. Attachment of spirochetes to the epithelial surface of the lower intestine is considered to be the hallmark of CS. However, because B. pilosicoli, B. aalborgi and unclassified flagellated bacteria are found singly or together in humans and non-human primates with CS lesions, attachment of spiral-shaped bacteria may not represent the same etiopathogenetic entity in all hosts. Moreover, North American opossums with CS are infected with B. aalborgi-like spirochetes together with flagellated bacteria, whereas B. pilosicoli is found alone in dogs, pigs, chickens and other species of birds with CS. Conversely, guinea-pigs with CS have unidentified spirochetes that may be B. pilosicoli or B. aalborgi. The pig model of CS suggests that attachment of B. pilosicoli to epithelial cells may be transient. By contrast, persistence of B. pilosicoli in the cecal and colonic crypt lumina, chronic inflammation caused by spirochetal invasion into the subepithelial lamina propria and translocation to extraintestinal sites may be more important than previously thought. This review describes the lesions seen in naturally occurring and experimentally induced CS of animals, and it sets the stage for future research into the pathogenic mechanisms of infection and colitis caused by B. pilosicoli.

Eradication of endemic Brachyspira pilosicoli infection from a farrowing herd: a case report.

Brachyspira pilosicoli and B. innocens were isolated repeatedly from a herd of 60 sows which mostly produced feeder pigs but also raised some fattening pigs. Postweaning diarrhea had been a severe problem in this herd for years. The B. pilosicoli eradication plan was based on the general guidelines for elimination of B. hyodysenteriae, with some modifications. The eradication measures were run in August 1997. In-feed medication with 200 p.p.m. tiamulin lasted for 18-30 days, depending on the age group. The piggery unit was emptied, cleaned, disinfected and dried, and all worn surfaces were repaired. The animals were removed to temporary sheds situated 0-100 m from the piggery unit. Only the sows and the boar returned to the piggery unit. All other pigs were sold from the sheds within 3 months after the eradication. Immediately after the eradication, the clinical postweaning diarrhea disappeared. The success of the program was monitored four times bacteriologically, and the last control sampling was in December 1999, 7 months after the total withdrawal of antimicrobial feed additives. The primary cultures from the last three samplings were also analysed with B. pilosicoli-specific PCR. All the samples were negative for B. pilosicoli. However, B. innocens could be isolated from each batch of samples. The analysis of B. innocens isolates by pulsed-field gel electrophoresis indicated that at least one genotype persisted in the herd. The clinical and laboratory findings suggest that the eradication of B. pilosicoli had succeeded in this herd.

Evaluation of blood culture systems for detection of the intestinal spirochaete Brachyspira (Serpulina) pilosicoli in human blood.

The anaerobic intestinal spirochaete Brachyspira (Serpulina) pilosicoli has been isolated from the bloodstream of French patients by manual blood culture systems. The purpose of this study was to determine whether the automated and manual blood culture systems used in Australia are suitable for growth and detection of this organism. Strains of B. pilosicoli were added to human blood to give concentrations ranging from 1 x 10(4) to 1 x 10(1) spirochaetes/ml and 10-ml volumes were inoculated into the media. Three strains of B. pilosicoli grew slowly in all manual Hemoline and BBL Septi-Chek formulations tested. Subcultures taken between 2 and 10 days after inoculation yielded growth only after incubation for a further 5-8 days. Growth and automated detection were achieved in the BACTEC system with Anaerobic/F medium with or without Fastidious Organism Supplement. Minimum time to signal for nine strains varied between 5.6 and 14.9 days, with a minimum concentration of 10(1) spirochaetes/ml of blood being detected. None of nine strains gave a positive signal in the BacT/Alert system when FAN Anaerobic culture bottles were used; however, four strains were detected by subculture taken at 7 or 14 days after inoculation. When Anaerobic medium was used in the BacT/Alert system, two of three strains gave a signal and the other strain grew and was detected by subculture. Spirochaetaemias caused by B. pilosicoli may be unrecognised because detection time by the signal or subculture exceeds 5 days.

Influences of diet and vaccination on colonisation of pigs by the intestinal spirochaete Brachyspira (Serpulina) pilosicoli.

The purpose of this study was to determine whether methods used to control swine dysentery (SD), caused by the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, would also be effective in controlling porcine intestinal spirochaetosis (PIS) caused by the related spirochaete Brachyspira (Serpulina) pilosicoli. Weaner pigs in Groups I (n=8) and II (n=6) received a standard weaner pig diet based on wheat and lupins, whilst Group III (n=6) received an experimental diet based on cooked white rice and animal protein. Pigs in Group II were vaccinated intramuscularly twice at a 3-week-interval with a formalinised bacterin made from B. pilosicoli porcine strain 95/1000 resuspended in Freund's incomplete adjuvant. Eleven days later pigs in all groups were infected orally with 10(10) cells of strain 95/1000 on three successive days. One control pig in Group I developed acute diarrhoea, and at post-mortem had a severe erosive colitis with end-on attachment of spirochaetes to the colonic epithelium. All other pigs developed transient mild diarrhoea and had moderate patchy colitis at post-mortem 3 weeks later. B. pilosicoli was isolated from the faeces of all pigs, except for one fed rice, and was isolated from the mesenteric nodes of three pigs from Group I and from one vaccinated pig in Group II. Consumption of the rice-based diet, but not vaccination, delayed and significantly (p<0.001) reduced the onset of faecal excretion of B. pilosicoli after experimental challenge. Vaccination induced a primary and secondary serological response to B. pilosicoli, as measured using sonicated whole cells of strain 95/1000 as an ELISA plate coating antigen. Antibody titres in the vaccinated pigs then declined, despite intestinal colonisation by B. pilosicoli. Both groups of unvaccinated animals also failed to develop a post-infection increase in circulating antibody titres.

Canine intestinal spirochetes consist of Serpulina pilosicoli and a newly identified group provisionally designated "Serpulina canis" sp. nov.

The spirochetes inhabiting the large intestines of humans and animals consist of a diverse group of related organisms. Intestinal spirochetosis caused by Serpulina pilosicoli is a newly recognized enteric disease of human beings and animals with potential public health significance. The purpose of this study was to determine the species identity of canine intestinal spirochetes by comparing 30 isolates obtained from dogs in Australia (n = 25) and the United States (n = 5) with reference strains representing Serpulina species and Brachyspira aalborgi, by phenotypic and genetically based typing methods. All of the canine isolates were indole negative and produced a weak beta-hemolysis when cultured anaerobically on agar medium containing blood. Four isolates were identified as S. pilosicoli by 16S rRNA-specific PCR assays, rRNA gene restriction fragment length polymorphism or ribotyping, and multilocus enzyme electrophoresis. The remaining 26 isolates formed a cluster related to porcine Serpulina innocens as determined by multilocus enzyme electrophoresis but had a unique ribotype pattern. The data suggested the existence of a novel Serpulina species, provisionally designated "Serpulina canis," colonizing the intestines of dogs.

Evaluation of day-old specific pathogen-free chicks as an experimental model for pathogenicity testing of intestinal spirochaete species.

Specific pathogen-free chicks aged 1 day were challenged per os with strains of five different species of intestinal spirochaete originally isolated from pigs or human beings. A virulent strain of Serpulina hyodysenteriae (WA 15) colonized chicks, causing retarded growth rate and histological changes, including caecal atrophy, epithelial and goblet cell hyperplasia, and crypt elongation. A further strain of S. hyodysenteriae (SA3), which was apparently avirulent for pigs, and a strain of Serpulina intermedia (889) colonized fewer chicks, caused less severe lesions and did not significantly depress growth rate. Strains of Serpulina murdochii and Brachyspira aalborgi failed to colonize or cause histological changes. Four strains of Serpulina pilosicoli (Kar, Rosie-2299 and GAP 401, isolated from human beings, and 3295, isolated from a pig) colonized chicks, and large numbers showed polar attachment to the caecal epithelium; all strains, apart from Rosie-2299, caused watery diarrhoea and wet litter, but did not significantly retard growth. Variation both in the degree of spirochaetal attachment and the resulting development of lesions was observed between S. pilosicoli strains as well as between individual chicks infected with the same strain. The study indicated that chicks may be useful in studying the pathogenicity of strains of S. hyodysenteriae, S. intermedia and S. pilosicoli.

Bacteriophages induced from weakly beta-haemolytic human intestinal spirochaetes by mitomycin C.

A comparative electron microscopic analysis of weakly beta-haemolytic spirochaetes related to human and animal intestinal spirochaetosis was done in order to search for the presence of inducible bacteriophages associated with these spirochaetes. Bacteriophages were detected at the electron microscope after experimental induction with mitomycin C in 4 strains of weakly beta-haemolytic spirochaetes related to human intestinal spirochaetosis, in Serpulina pilosicoli strain P43/6/78, the causative agent of swine intestinal spirochaetosis, in a spirochaetal strain related to avian intestinal spirochaetosis, and in Serpulina hyodysenteriae, strain P18A, the causative agent of swine dysentery, which was comparatively analysed as control. All phage-particles observed in both human and animal intestinal spirochaetes were morphologically similar with an isometric head of 45 nm diameter and a tail 63-70 nm long and 7-12 nm width. The presence of morphologically similar phages in all the haemolytic intestinal spirochaetes of human and animal origin analysed in this study opens some important questions, about the genetic relationship of phages present in pathogenic intestinal spirochaetes, their host range, and the possibility of natural gene transfer among pathogenic haemolytic intestinal spirochaetes of human and animal origin.