Histologic characteristics of human intestinal spirochetosis in operatively resected specimens.

Human intestinal spirochetosis (HIS), one of the zoonoses, is caused by colonization by Brachyspira species bacteria within the large intestine. Histologic diagnosis of HIS is usually established by finding "fringes" on the colonic surface epithelium in biopsy specimens. However, its histologic characteristics, especially beneath the colonic mucosa, have not been elucidated. The present study was designed to examine the histologic characteristics of HIS in operatively resected specimens. We reviewed operatively resected (colectomy or appendectomy) specimens obtained in six consecutive years at a single medical center. HIS was diagnosed histologically by finding "fringes". Immunohistochemical study using anti-Treponema pallidum antibody, which cross-reacts with Brachyspira, was additionally performed. A total of 848 (M:F = 477:371; median age, 59 years; 12-94 years) colectomy and/or appendectomy cases were examined, and the seven cases (0.8%) diagnosed as having HIS were all male (1.5% of male cases). Four HIS cases (0.8% of 508 colectomy cases (1.4% of 285 male-cases)) were colectomy cases with cancers, and the other three (0.9% of 340 appendectomy cases (1.6% of 192 male-cases)) were appendectomy cases for acute appendicitis. Our study revealed (1) a heterogeneous distribution of diagnostically important "fringes" within the large intestine, (2) an ileal presence of Brachyspira, (3) superficial location of HIS-related findings among anatomical wall layers, and (4) the presence of Brachyspira or its derivatives within macrophages in the lamina propria and immune apparatus (lymphoid follicles in superficial wall structures (lamina propria or submucosa) and lymph nodes). Investigation using operatively resected specimens might help elucidate the characteristics of HIS. Brachyspira may have immunogenicity in humans.

Hiding in Plain Sight: Colonic Spirochetosis in Humans.

In 1967, Harland and Lee made a startling discovery: in some humans, the colonic epithelium is covered with a "forest" of spirochetes (W. A. Harlan, and F. D. Lee, Br Med J 3:718-719, 1967, https://doi.org/10.1136/bmj.3.5567.718). In this issue of Journal of Bacteriology, Thorell et al. present a systematic analysis of the prevalence and diversity of the spirochetes Brachyspira aalborgi and Brachyspira pilosicoli in the human colon. These and prior studies provide avenues toward resolving important questions: what bacterial and host parameters contribute to this extensive colonization, and what impact does it have on human health?

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.

Immunohistochemical study of mucins in human intestinal spirochetosis.

Most patients with human intestinal spirochetosis (HIS; a colorectal bacterial infection caused by Brachyspira species) seem asymptomatic, and its pathogenicity remains unclear. Recently, alterations in mucin expression were reported in animal Brachyspira infection. The present question was "Is mucin expression altered in HIS?" Using antibodies for MUCs 1, 2, 4, 5AC, and 6, we immunohistochemically compared 215 specimens from 83 histology-confirmed HIS cases with 106 specimens from 26 non-HIS cases. Positive staining (which included even focal positive staining) was rated "high (+)" or "low (+)." Results were analyzed for 4 categories of lesions, and associations between MUC expression and spirochetal presence were also analyzed. In the "specimens without polyps or adenocarcinoma" category, high (+) MUC2 positivity was more frequent in HIS than in control. In the hyperplasia/serrated polyp category, in HIS (versus control), the MUC5AC positivity rate was lower, whereas high (+) MUC4 positivity was more frequent. In the conventional adenoma category, in HIS (versus control), the MUC1 positivity rate was lower, whereas both high (+) MUC2 positivity and high (+) MUC5AC positivity were less frequent. In the adenocarcinoma category, high (+) MUC2 positivity was more frequent in HIS than in control. Among the above mucins, only MUC1 positivity was significantly associated with an absence of the so-called fringe formation, an absence of spiral organisms within mucus, and an absence of strong immunopositive materials within the epithelial layer and within the subepithelial layer. The results suggest that Brachyspira infection or a related change in the microbiome may alter the large intestine mucin expression profile in humans.

An Investigation into the Etiological Agents of Swine Dysentery in Australian Pig Herds.

Swine dysentery (SD) is a mucohemorrhagic colitis, classically seen in grower/finisher pigs and caused by infection with the anaerobic intestinal spirochete Brachyspira hyodysenteriae. More recently, however, the newly described species Brachyspira hampsonii and Brachyspira suanatina have been identified as causing SD in North America and/or Europe. Furthermore, there have been occasions where strains of B. hyodysenteriae have been recovered from healthy pigs, including in multiplier herds with high health status. This study investigated whether cases of SD in Australia may be caused by the newly described species; how isolates of B. hyodysenteriae recovered from healthy herds compared to isolates from herds with disease; and how contemporary isolates compare to those recovered in previous decades, including in their plasmid gene content and antimicrobial resistance profiles. In total 1103 fecal and colon samples from pigs in 97 Australian herds were collected and tested. Of the agents of SD only B. hyodysenteriae was found, being present in 34 (35.1%) of the herds, including in 14 of 24 (58%) herds that had been considered to be free of SD. Multilocus sequence typing applied to 96 isolates from 30 herds and to 53 Australian isolates dating from the 1980s through the early 2000s showed that they were diverse, distinct from those reported in other countries, and that the 2014/16 isolates generally were different from those from earlier decades. These findings provided evidence for ongoing evolution of B. hyodysenteriae strains in Australia. In seven of the 20 herds where multiple isolates were available, two to four different sequence types (STs) were identified. Isolates with the same STs also were found in some herds with epidemiological links. Analysis of a block of six plasmid virulence-associated genes showed a lack of consistency between their presence or absence and their origin from herds currently with or without disease; however, significantly fewer isolates from the 2000s and from 2014/16 had this block of genes compared to isolates from the 1980s and 1990s. It is speculated that loss of these genes may have been responsible for the occurrence of milder disease occurring in recent years. In addition, fewer isolates from 2014/16 were susceptible to the antimicrobials lincomycin, and to a lesser extent tiamulin, than those from earlier Australian studies. Four distinct multi-drug resistant strains were identified in five herds, posing a threat to disease control.

Investigation of the impact of increased dietary insoluble fiber through the feeding of distillers dried grains with solubles (DDGS) on the incidence and severity of Brachyspira-associated colitis in pigs.

Diet has been implicated as a major factor impacting clinical disease expression of swine dysentery and Brachyspira hyodysenteriae colonization. However, the impact of diet on novel pathogenic strongly beta-hemolytic Brachyspira spp. including "B. hampsonii" has yet to be investigated. In recent years, distillers dried grains with solubles (DDGS), a source of insoluble dietary fiber, has been increasingly included in diets of swine. A randomized complete block experiment was used to examine the effect of increased dietary fiber through the feeding of DDGS on the incidence of Brachyspira-associated colitis in pigs. One hundred 4-week-old pigs were divided into five groups based upon inocula (negative control, Brachyspira intermedia, Brachyspira pilosicoli, B. hyodysenteriae or "B. hampsonii") and fed one of two diets containing no (diet 1) or 30% (diet 2) DDGS. The average days to first positive culture and days post inoculation to the onset of clinical dysentery in the B. hyodysenteriae groups was significantly shorter for diet 2 when compared to diet 1 (P = 0.04 and P = 0.0009, respectively). A similar difference in the average days to first positive culture and days post inoculation to the onset of clinical dysentery was found when comparing the "B. hampsonii" groups. In this study, pigs receiving 30% DDGS shed on average one day prior to and developed swine dysentery nearly twice as fast as pigs receiving 0% DDGS. Accordingly, these data suggest a reduction in insoluble fiber through reducing or eliminating DDGS in swine rations should be considered an integral part of any effective disease elimination strategy for swine dysentery.

Cessation of clinical disease and spirochete shedding after tiamulin treatment in pigs experimentally infected with "Brachyspira hampsonii".

With the emergence of "Brachyspira hampsonii" associated with swine dysentery in North America, identification of effective treatments and interventions is a pressing need. Denagard® (tiamulin hydrogen fumarate) Liquid Concentrate 12.5% is approved in the United States for treatment of dysentery caused by Brachyspira hyodysenteriae at 0.006% in the water. In this study, the effectiveness of tiamulin in resolving clinical disease, eliminating viable spirochete shedding, and reducing neutrophilic colitis following infection with either "B. hampsonii" or B. hyodysenteriae was evaluated. Seventy-eight 7-week-old crossbred pigs were divided into three groups [sham-inoculated (n = 18), "B. hampsonii"-inoculated (n = 30), and B. hyodysenteriae-inoculated (n = 30)]. Each inoculum group was divided into three subgroups which received either 0.006% tiamulin, 0.018% tiamulin, or no medication. Both levels of tiamulin resolved clinical disease within 24 h of treatment initiation, eliminated spirochete shedding within 72 h of treatment initiation, and resolved and/or prevented histologic lesions in pigs infected with either Brachyspira spp.

Comparison of sesion severity, distribution, and colonic mucin expression in pigs with acute swine dysentery following oral inoculation with "Brachyspira hampsonii" or Brachyspira hyodysenteriae.

Swine dysentery is classically associated with infection by Brachyspira hyodysenteriae, the only current officially recognized Brachyspira sp. that consistently imparts strong beta-hemolysis on blood agar. Recently, several strongly beta-hemolytic Brachyspira have been isolated from swine with clinical dysentery that are not identified as B. hyodysenteriae by PCR including the recently proposed species "Brachyspira hampsonii." In this study, 6-week-old pigs were inoculated with either a clinical isolate of "B. hampsonii" (EB107; n = 10) clade II or a classic strain of B. hyodysenteriae (B204; n = 10) to compare gross and microscopic lesions and alterations in colonic mucin expression in pigs with clinical disease versus controls (n = 6). Gross lesions were similar between infected groups. No histologic difference was observed between infected groups with regard to neutrophilic inflammation, colonic crypt depth, mucosal ulceration, or hemorrhage. Histochemical and immunohistochemical evaluation of the apex of the spiral colon revealed decreased expression of sulphated mucins, decreased expression of MUC4, and increased expression of MUC5AC in diseased pigs compared to controls. No difference was observed between diseased pigs in inoculated groups. This study reveals significant alterations in colonic mucin expression in pigs with acute swine dysentery and further reveals that these and other microscopic changes are similar following infection with "B. hampsonii" clade II or B. hyodysenteriae.

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.

Evidence for systemic spread of the potentially zoonotic intestinal spirochaete Brachyspira pilosicoli in experimentally challenged laying chickens.

Brachyspira pilosicoli is a potentially zoonotic anaerobic intestinal spirochaete that is one of several species causing avian intestinal spirochaetosis. The aim of this study was to develop a reproducible model of infection in point-of-lay chickens and compare the virulence of two strains of B. pilosicoli in a model using experimentally challenged laying chickens. Seventeen-week-old commercial laying chickens were experimentally challenged by oral gavage with either B. pilosicoli strain B2904 or CPSp1, following an oral dose of 10 % sodium bicarbonate to neutralize acidity in the crop. Approximately 80 % of the chickens became colonized and exhibited increased faecal moisture content, reduced weight gain and delayed onset of lay. Tissues sampled at post-mortem examination were analysed to produce a quantitative output on the number of spirochaetes present and hence, the extent of colonization. The liver and spleen were colonized, and novel histopathology was observed in these tissues. The infection model we report here has potential use in studies to improve our understanding of the mechanisms by which Brachyspira elicit disease in poultry and in testing novel intervention strategies.

Comparative virulence of clinical Brachyspira spp. isolates in inoculated pigs.

Classical swine dysentery is associated with the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae. However, multiple Brachyspira spp. can colonize the porcine colon. Since 2008, several Brachyspira spp. not identified as B. hyodysenteriae by genotypic and/or phenotypic methods have been isolated from the feces of pigs with clinical disease typical of swine dysentery. In the current study, 8 clinical isolates, including 5 strongly beta-hemolytic and 3 weakly beta-hemolytic Brachyspira strains, and a reference strain of B. hyodysenteriae (B204) were inoculated into pigs (n = 6 per isolate) to compare pathogenic potential following oral inoculation. Results revealed that strongly beta-hemolytic isolates induced significantly greater typhlocolitis than those that are weakly beta-hemolytic, regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as "Brachyspira sp. SASK30446" and Brachyspira intermedia by polymerase chain reaction (PCR) produced lesions similar to those caused by B. hyodysenteriae. The results suggest that phenotypic culture characteristics of Brachyspira spp. may be a more sensitive indicator of potential to induce dysentery-like disease in pigs than molecular identification alone based on currently available PCR assays. Additionally, culture of mucosal scrapings obtained at necropsy was more sensitive than direct PCR on the same samples for detection of Brachyspira spp.

Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery.

Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as 'Brachyspira sp. SASK30446' and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model.

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

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

Experimental challenge of mallards (Anas platyrhynchos) with Brachyspira hyodysenteriae and "Brachyspira suanatina" isolated from pigs and mallards.

Brachyspira hyodysenteriae, the aetiological agent of swine dysentery, and a recently proposed and closely related enteropathogenic spirochaete "Brachyspira suanatina", originally isolated from pigs or mallards (Anas platyrhynchos), were used to inoculate week-old mallard ducklings orally or cloacally. The colonization rate, clinical outcome, faecal dry matter content, blood leucocyte counts and gross, microscopical and electron microscopical features 14-16 days post-inoculation were investigated at necropsy examination. Strains of "B. suanatina" of pig and mallard origin and B. hyodysenteriae of mallard origin colonized the ducklings by oral inoculation, and colonization was also established by cloacal inoculation with a "B. suanatina" strain of mallard origin. The porcine reference strain of B. hyodysenteriae (B204) failed to colonize the birds. Unchallenged contact birds in one of the challenge groups were readily colonized by a strain of "B. suanatina" of mallard origin. The proportion of colonized birds differed significantly between the challenge groups (P < 0.0001). For each challenge group, the inoculum and a randomly selected subset of recovered isolates had an identical biochemical profile and banding pattern by randomly amplified polymorphic DNA (RAPD) analysis. None of the birds developed clinical signs of gastrointestinal disease during the trial. The faecal dry weight contents, body weights and total leucocyte and heterophil counts did not differ between the various groups of birds. At the microscopical and electron microscopical levels, the caecal mucosa in some of the Brachyspira culture-positive birds had sharply demarcated epithelial cell changes and there were features of irreversible cell damage in crypt necks coinciding with spirochaetal infiltration of the mucosa. The crypts in Brachyspira culture-positive birds were deeper than in culture-negative birds (median: 237 microm and 218 microm, respectively, P = 0.019). This challenge model was well suited for use in mallards and consistent with previous findings that strongly haemolytic Brachyspira spp. may cross the species barrier between pigs and birds.

In vitro antagonistic activities of Lactobacillus spp. against Brachyspira hyodysenteriae and Brachyspira pilosicoli.

The sensitivity of Brachyspira hyodysenteriae and Brachyspira pilosicoli, respectively the causative agents of Swine Dysentery and Porcine Intestinal Spirochaetosis to two probiotic Lactobacillus strains, L. rhamnosus CNCM-I-3698 and L. farciminis CNCM-I-3699 was studied through viability, motility and coaggregation assays. The cell-free supernatant of these lactobacilli contains lactic acid, that is stressful for Brachyspira (leading to the formation of spherical bodies), and lethal. It was demonstrated for the first time the in vitro coaggregation properties of two probiotic Lactobacillus strains (active or heat-treated) with two pathogenic strains of Brachyspira, leading to (1) trapping of spirochaetal cells in a physical network as demonstrated by SEM; (2) inhibition of the motility of Brachyspira. Such in vitro studies should encourage in vivo studies in animal model to evaluate the potential of the use of probiotic lactobacilli through a feeding strategy for the prevention of B. hyodysenteriae and B. pilosicoli.

Experimental study on the role of Brachyspira alvinipulli in intestinal spirochaetosis of geese.

Ten one-day-old goslings were inoculated orally with a Brachyspira alvinipulli strain isolated from the large intestine of geese that had died of intestinal spirochaetosis (Group A), 10 day-old goslings were inoculated orally with a B. hyodysenteriae strain (Group B), and a third group of 10 goslings (Group C) served as uninfected control. The goslings were observed daily for clinical signs. They were sacrificed on days 7, 14, 21 and 35 days postinfection (PI), and necropsied. Segments of the large intestine were subjected to histopathological, immunohistochemical, electron microscopic (TEM, SEM) and microbiological examinations. Mortality did not occur during the experimental period. However, in both groups the caecum of the goslings killed by bleeding was slightly dilated, in its lumen there was a watery, yellowish and frothy content, and the mucous membrane was slightly swollen. By histopathological, immunohistochemical and electron microscopic examination, B. alvinipulli and B. hyodysenteriae could be detected in the caecum or colon, in the lumen of the glands and sometimes among the glandular epithelial cells in goslings of the respective groups, and could be reisolated from these organs by culturing. A mild inflammation of the intestinal mucosa was also noted. In transverse section of the brachyspirae, numerous (16-22) periplasmic flagella could be detected inside the outer sheath, also depending on the plane of section.

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.

Neither hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli type strain caused diarrhoea in early-weaned pigs by experimental infection.

A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.