Brachyspira in dogs: risk factors of shedding in central Germany and longitudinal study of an infected kennel.

BACKGROUND: Brachyspira (B.) pilosicoli is a zoonotic pathogen, able to infect different animal species such as pigs, poultry, and rodents, causing intestinal spirochetosis. An association of gastrointestinal clinical signs, such as diarrhea, with the isolation of B. pilosicoli from fecal samples or rectal swabs has not been proven in dogs. Other Brachyspira species commonly isolated from dogs, such as "B. canis" and "B. pulli", are considered commensals. This study investigated the occurrence of different Brachyspira species in rectal swabs and fecal samples in an independent canine cohort in central Germany. These included samples from shelter dogs, hunting dogs, and dogs presenting at regional small animal practices with various clinical signs. Data about the dogs, including potential risk factors for Brachyspira isolation, were obtained using a standardized questionnaire. The study also longitudinally investigated a colony of Beagle dogs for Brachyspira over 5 years. RESULTS: The rate of Brachyspira spp. isolation was 11% and included different Brachyspira species ("B. canis", "B. pulli", and B. pilosicoli). "B. canis" was detected in 18 dogs, whereas B. pilosicoli was only isolated from 1 dog in the independent cohort (not including the Beagle colony). Risk factors for shedding Brachyspira and "B. canis" were being less than 1 year of age and shelter origin. Gastrointestinal signs were not associated with the shedding of Brachyspira. B. pilosicoli and "B. canis" were isolated from several dogs of the same Beagle colony in 2017 and again in 2022, while Brachyspira was not isolated at multiple sampling time points in 2021. CONCLUSIONS: Shedding of B. pilosicoli in dogs appears to be uncommon in central Germany, suggesting a low risk of zoonotic transmission from dogs. Commensal status of "B. canis" and "B. pulli" is supported by the results of this study. Findings from the longitudinal investigation of the Beagle colony agree with an asymptomatic long-term colonization of dogs with "B. canis" and B. pilosicoli and suggest that introducing new animals in a pack can trigger an increased shedding of B. pilosicoli.

First molecular detection of Brachyspira hampsonii on pig farms in Poland.

Nowadays, the three strongly beta-haemolytic spirochaetes, Brachyspira hyodysenteriae, Brachyspira suanatina and Brachyspira hampsonii are thought to be causative agents of swine dysentery, an economically devastating disease of grow-finish pigs characterised by severe mucohaemorrhagic diarrhoea. B. hyodysenteriae has been reported in most leading swine-producing regions. B. suanatina and B. hampsonii have been successfully recovered from faecal samples collected in a few countries only. The present study was performed in March 2023 on faecal samples originating from nine Polish finisher farms with 6,000 to 18,000 animals in a location. Samples were obtained from 40 diarrhoeic finishers. Nucleic acid extracted from the samples was analysed using multiplex PCR for Brachyspira spp. From a total of nine sample populations examined in our study, the genetic material of B. hampsonii was identified in two. To the best of our knowledge, this is the first report on molecular detection of B. hampsonii on pig farms outside North America, Belgium and Germany. Our research highlights the need for increased focus directed on laboratory testing strategies, the lack of which may perplex swine practitioners and severely hinder a definite diagnosis.

Improving the consistency of experimental swine dysentery inoculation strategies.

Swine dysentery (SD) caused by pathogenic Brachyspira spp. is an economic challenge for the swine industry. In research settings, experimental reproduction of swine dysentery typically relies on intragastric inoculation which has shown variable success. This project aimed to improve the consistency of the experimental inoculation protocol used for swine dysentery in our laboratory. Over six experiments, we evaluated the influence of group housing in inoculated pigs using a frozen-thawed broth culture of strongly hemolytic B. hyodysenteriae strain D19 (Trial A), compared the relative virulence of B. hyodysenteriae strains D19 and G44 (Trial B), compared inoculum volumes (50 mL vs 100 mL) for G44 and B. hampsonii 30446 (Trial C), and performed three independent trials evaluating intragastric inoculation using different oral inoculation methods: oral feed balls (Trial D), and oral syringe bolus of 100 mL (Trial E) or 300 mL (Trial F). Intragastric inoculation with a fresh broth culture of B. hyodysenteriae strain G44 resulted in a shorter incubation period and a higher proportionate duration of mucohemorrhagic diarrhea (MMHD) compared to D19. Intragastric inoculation with either 50 or 100 mL of B. hampsonii 30446 or B. hyodysenteriae (G44) were statistically equivalent. Oral inoculation with 100 mL or 300 mL also yielded similar results to intragastric inoculation but was more expensive due to the additional work and supplies associated with syringe training. Our future research will use intragastric inoculation with 100 mL of a fresh broth culture containing B. hyodysenteriae strain G44 as it yields a high incidence of mucohaemorrhagic diarrhea with a reasonable cost.

Whole-genome analyses reveal a novel prophage and cgSNPs-derived sublineages of Brachyspira hyodysenteriae ST196.

BACKGROUND: Brachyspira (B.) hyodysenteriae is a fastidious anaerobe spirochete that can cause swine dysentery, a severe mucohaemorragic colitis that affects pig production and animal welfare worldwide. In Switzerland, the population of B. hyodysenteriae is characterized by the predominance of macrolide-lincosamide-resistant B. hyodysenteriae isolates of sequence type (ST) ST196, prompting us to obtain deeper insights into the genomic structure and variability of ST196 using pangenome and whole genome variant analyses. RESULTS: The draft genome of 14 B. hyodysenteriae isolates of ST196, sampled during a 7-year period from geographically distant pig herds, was obtained by whole-genome sequencing (WGS) and compared to the complete genome of the B. hyodysenteriae isolate Bh743-7 of ST196 used as reference. Variability results revealed the existence of 30 to 52 single nucleotide polymorphisms (SNPs), resulting in eight sublineages of ST196. The pangenome analysis led to the identification of a novel prophage, pphBhCH20, of the Siphoviridae family in a single isolate of ST196, which suggests that horizontal gene transfer events may drive changes in genomic structure. CONCLUSIONS: This study contributes to the catalogue of publicly available genomes and provides relevant bioinformatic tools and information for further comparative genomic analyses for B. hyodysenteriae. It reveals that Swiss B. hyodysenteriae isolates of the same ST may have evolved independently over time by point mutations and acquisition of larger genetic elements. In line with this, the third type of mobile genetic element described so far in B. hyodysenteriae, the novel prophage pphBhCH20, has been identified in a single isolate of B. hyodysenteriae of ST196.

Association between Brachyspira and irritable bowel syndrome with diarrhoea.

OBJECTIVE: The incidence of IBS increases following enteric infections, suggesting a causative role for microbial imbalance. However, analyses of faecal microbiota have not demonstrated consistent alterations. Here, we used metaproteomics to investigate potential associations between mucus-resident microbiota and IBS symptoms. DESIGN: Mucus samples were prospectively collected from sigmoid colon biopsies from patients with IBS and healthy volunteers, and their microbial protein composition analysed by mass spectrometry. Observations were verified by immunofluorescence, electron microscopy and real-time PCR, further confirmed in a second cohort, and correlated with comprehensive profiling of clinical characteristics and mucosal immune responses. RESULTS: Metaproteomic analysis of colon mucus samples identified peptides from potentially pathogenic Brachyspira species in a subset of patients with IBS. Using multiple diagnostic methods, mucosal Brachyspira colonisation was detected in a total of 19/62 (31%) patients with IBS from two prospective cohorts, versus 0/31 healthy volunteers (p<0.001). The prevalence of Brachyspira colonisation in IBS with diarrhoea (IBS-D) was 40% in both cohorts (p=0.02 and p=0.006 vs controls). Brachyspira attachment to the colonocyte apical membrane was observed in 20% of patients with IBS and associated with accelerated oro-anal transit, mild mucosal inflammation, mast cell activation and alterations of molecular pathways linked to bacterial uptake and ion-fluid homeostasis. Metronidazole treatment paradoxically promoted Brachyspira relocation into goblet cell secretory granules-possibly representing a novel bacterial strategy to evade antibiotics. CONCLUSION: Mucosal Brachyspira colonisation was significantly more common in IBS and associated with distinctive clinical, histological and molecular characteristics. Our observations suggest a role for Brachyspira in the pathogenesis of IBS, particularly IBS-D.

Brachyspira Species Avidity to Colonic Mucins from Pigs with and without Brachyspira hyodysenteriae Infection Is Species Specific and Varies between Strains.

Brachyspira hyodysenteriae is commonly associated with swine dysentery (SD), a disease that has an economic impact on the swine industry. B. hyodysenteriae infection results in changes to the colonic mucus niche with massive mucus induction, which substantially increases the number of B. hyodysenteriae binding sites in the mucus. We previously determined that a B. hyodysenteriae strain binds to colon mucins in a manner that differs between pigs and mucin types. Here, we investigated if adhesion to mucins is a trait observed across a broad set of B. hyodysenteriae strains and isolates and furthermore at a genus level (B. innocens, B. pilosicoli, B. murdochii, B. hampsonii, and B. intermedia strains). Our results show that binding to mucins appears to be specific to B. hyodysenteriae, and within this species, the binding ability to mucins varies between strains/isolates, increases for mucins from pigs with SD, and is associated with sialic acid epitopes on mucins. Infection with B. hyodysenteriae strain 8dII results in mucin glycosylation changes in the colon, including a shift in sialic acid-containing structures. Thus, we demonstrate through hierarchical cluster analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) models of the relative abundances of sialic acid-containing glycans that sialic acid-containing structures in the mucin O-glycome are good predictors of B. hyodysenteriae strain 8dII infection in pigs. The results emphasize the role of sialic acids in governing B. hyodysenteriae interactions with its host, which may open perspectives for therapeutic strategies.

Mucosal eosinophilic infiltration may be a characteristic of human intestinal spirochetosis.

BACKGROUND: Human intestinal spirochetosis (HIS) is an infectious disease of large intestines caused by Brachyspira species, and most HIS cases are asymptomatic or exhibit mild intestinal symptoms. The host reaction to HIS remains unclear, and we examined HIS-related mucosal inflammatory features histologically. METHODS: From the archival HIS cases in a single medical center, 24 endoscopically taken specimens from 14 HIS cases (male:female = 10:4; 28-73 yrs) were selected as not containing polypoid or neoplastic lesions. Stromal neutrophils, eosinophils, and mast cells, and intraepithelial neutrophils and eosinophils, (sNeu, sEo, sMast, iNeu, and iEo, respectively) were counted, and the presence or absence of lymphoid follicles/aggregates (LFs) was also examined. Association of the above inflammation parameters and spirochetal infection parameters (such as degrees of characteristic fringe distribution, of spirochetal cryptal invasion, and of spirochetal intraepithelial invasion) were also analysed. RESULTS: iNeu was observed in 29.2%, iEo in 58.3%, and LFs in 50.0% of the specimens. Maximal counts of sNeu, sEo, sMast, iNeu, and iEo averaged 8.4, 21.5, 6.0, 0.5 and 1.5, respectively. Strong correlation between the maximum counts of iNeu and iEo (p < 0.001, r = 0.81), and correlations between those of iEo and sNeu (p = 0.0012, r = 0.62) and between those of iEo and sEo (p = 0.026, r = 0.45) were observed. iNeu was influenced by fringe formation (p < 0.05) and spirochetal crypt involvement (p < 0.05). CONCLUSIONS: HIS was accompanied by inflammatory reactions, and among these, mucosal eosinophilic infiltration may be a central indicator and host reaction of HIS.

Impairment of electroneutral Na+ transport and associated downregulation of NHE3 contributes to the development of diarrhea following in vivo challenge with Brachyspira spp.

The effect of Brachyspira hyodysenteriae and Brachyspira hampsonii spirochetosis on Na+ transport was assessed in the colon to determine its contribution to diarrheal disease in pigs following experimental infection. Electrogenic and electroneutral Na+ absorption was assessed in Ussing chambers by radiolabeled 22Na flux and pharmacological inhibitory studies. Basal radiolabeled 22Na flux experiments revealed that mucosal-to-serosal flux (Jms) was significantly impaired in B. hyodysenteriae and B. hampsonii-diseased pigs. Inhibition of epithelial sodium channel via amiloride did not significantly reduce electrogenic short-circuit current (Isc) in the proximal, apex, and distal colonic segments of diseased pigs over control pigs, suggesting that a loss of electroneutral Na+ absorption is responsible for diarrheal development. These findings were further supported by significant downregulation of Na+/H+ exchanger (NHE1, NHE2, and NHE3) mRNA expression in the proximal, apex, and distal colonic segments paired with decreased protein expression of the critical NHE3 isoform. The decrease in NHE3 mRNA expression appears not to be attributed to the host's cytokine response as human IL-1α did not modify NHE3 mRNA expression in Caco-2 cells. However, a whole cell B. hampsonii lysate significantly downregulated NHE3 mRNA expression and significantly increased p38 phosphorylation in Caco-2 cells. Together these findings provide a likely mechanism for the spirochete-induced malabsorptive diarrhea, indicated by a decrease in electroneutral Na+ absorption in the porcine colon due to Brachyspira's ability to inhibit NHE3 transcription, resulting in diarrheal disease.NEW & NOTEWORTHY This research demonstrates that diarrheal disease caused by two infectious spirochete spp. is a result of impaired electroneutral Na+ absorption via Na+/H+ exchanger 3 (NHE3) in the porcine colon. Our findings suggest that the decrease in NHE3 mRNA and protein is not likely a result of the host's cytokine response. Rather, it appears that these two Brachyspira spp. directly inhibit the transcription and translation of NHE3, resulting in the development of diarrhea.

Swine dysentery disease mechanism: Brachyspira hampsonii impairs the colonic immune and epithelial repair responses to induce lesions.

Swine dysentery (SD) is a global, production-limiting disease of pigs in commercial farms. It is associated with infection by Brachyspira hyodysenteriae and B. hampsonii, and characterized by mucohaemorrhagic diarrhea and colitis, SD prevention, treatment or control relies heavily on antimicrobials as no commercial vaccines are available. This is linked to our poor understanding of the disease pathogenesis. Our goal was to characterize the host-pathogen interactions during the early stage of infection. We employed dual RNA-seq to profile mRNA and miRNA following 1-h incubation of colonic explants with a pathogenic or a non-pathogenic B. hampsonii strain. Our results suggest that the pathogenic strain more efficiently interfered with the host's ability to activate and build a humoral response (through IL-4/CCR6/KLHL6 interactions), epithelial wound repair mechanisms (associated with LSECtin impairment of macrophages), induced mitochondrial dysfunction (linked to MDR1), and loss of microbiome homeostasis. The pathogenic strain also up-regulated the expression of stress-associated genes, when compared to the non-pathogenic strain. These results shed a light on the pathophysiological mechanisms that lead to SD and will contribute to the development of novel disease control tools.

The Spirochete Brachyspira pilosicoli, Enteric Pathogen of Animals and Humans.

Brachyspira pilosicoli is a slow-growing anaerobic spirochete that colonizes the large intestine. Colonization occurs commonly in pigs and adult chickens, causing colitis/typhlitis, diarrhea, poor growth rates, and reduced production. Colonization of humans also is common in some populations (individuals living in village and peri-urban settings in developing countries, recent immigrants from developing countries, homosexual males, and HIV-positive patients), but the spirochete rarely is investigated as a potential human enteric pathogen. In part this is due to its slow growth and specialized growth requirements, meaning that it is not detectable in human fecal samples using routine diagnostic methods. Nevertheless, it has been identified histologically attached to the colon and rectum in patients with conditions such as chronic diarrhea, rectal bleeding, and/or nonspecific abdominal discomfort, and one survey of Australian Aboriginal children showed that colonization was significantly associated with failure to thrive. B. pilosicoli has been detected in the bloodstream of elderly patients or individuals with chronic conditions such as alcoholism and malignancies. This review describes the spirochete and associated diseases. It aims to encourage clinicians and clinical microbiologists to consider B. pilosicoli in their differential diagnoses and to develop and use appropriate diagnostic protocols to identify the spirochete in clinical specimens.

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?

Isolates from Colonic Spirochetosis in Humans Show High Genomic Divergence and Potential Pathogenic Features but Are Not Detected Using Standard Primers for the Human Microbiota.

Colonic spirochetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance, and only a few bacterial isolates from this condition have been characterized to date. In a randomized, population-based study in Stockholm, Sweden, 745 healthy individuals underwent colonoscopy with biopsy sampling. Of these individuals, 17 (2.3%) had colonic spirochetosis, which was associated with eosinophilic infiltration and a 3-fold-increased risk for irritable bowel syndrome (IBS). We aimed to culture the bacteria and perform whole-genome sequencing of the isolates from this unique representative population sample. From 14 out of 17 individuals with spirochetosis we successfully isolated, cultured, and performed whole-genome sequencing of in total 17 isolates, including the Brachyspira aalborgi type strain, 513A. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and nonspirochetosis controls. We found one isolate to be of the species Brachyspira pilosicoli; all remaining isolates were of the species Brachyspira aalborgi Besides displaying extensive genetic heterogeneity, the isolates harbored several mucin-degrading enzymes and other virulence-associated genes that could confer a pathogenic potential in the human colon. We also showed that 16S amplicon sequencing using standard primers for human microbiota studies failed to detect Brachyspira due to primer incompatibility.IMPORTANCE This is the first report of whole-genome analysis of clinical isolates from individuals with colonic spirochetosis. This characterization provides new opportunities in understanding the physiology and potentials of these bacteria that densely colonize the gut in the individuals infected. The observation that standard 16S amplicon primers fail to detect colonic spirochetosis may have major implications for studies searching for associations between members of the microbiota and clinical conditions such as irritable bowel syndrome (IBS) and should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.

Exposure to UV-B Radiation Leads to Increased Deposition of Cell Wall-Associated Xerocomic Acid in Cultures of Serpula himantioides.

Many fungi are thought to have developed morphological and physiological adaptations to cope with exposure to UV-B radiation, but in most species, such responses and their protective effects have not been explored. Here, we study the adaptive response to UV-B radiation in the widespread, saprotrophic fungus Serpula himantioides, frequently found colonizing coniferous wood in nature. We report the morphological and chemical responses of S. himantioides to controlled intensities of UV-B radiation, under in vitro culture conditions. Ultraviolet radiation induced a decrease in the growth rate of S. himantioides but did not cause gross morphological changes. Instead, we observed accumulation of pigments near the cell wall with increasing intensities of UV-B radiation. Nuclear magnetic resonance (NMR) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) analyses revealed that xerocomic acid was the main pigment present, both before and after UV-B exposure, increasing from 7 mg/liter to 15 mg/liter after exposure. We show that xerocomic acid is a photoprotective metabolite with strong antioxidant abilities, as evidenced by DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], and oxygen radical absorbance capacity (ORAC) assays. Finally, we assessed the capacity of xerocomic acid as a photoprotective agent on HEK293 cells and observed better photoprotective properties than those of ß-carotene. Xerocomic acid is therefore a promising natural product for development as a UV-protective ingredient in cosmetic and pharmaceutical products.IMPORTANCE Our study shows the morphological and chemical responses of S. himantioides to controlled doses of UV-B radiation under in vitro culture conditions. We found that increased biosynthesis of xerocomic acid was the main strategy adopted by S. himantioides against UV-B radiation. Xerocomic acid showed strong antioxidant and photoprotective abilities, which has not previously been reported. Our results indicate that upon UV-B exposure, S. himantioides decreases its hyphal growth rate and uses this energy instead to increase the biosynthesis of xerocomic acid, which is allocated near the cell wall. This metabolic switch likely allows xerocomic acid to efficiently defend S. himantioides from UV radiation through its antioxidant and photoprotective properties. The findings further suggest that xerocomic acid is a promising candidate for development as a cosmetic ingredient to protect against UV radiation and should therefore be investigated in depth in the near future both in vitro and in vivo.

Colonic Spirochetes: What Has Genomics Taught Us?

The 'colonic' spirochetes assigned to the genus Brachyspira are slow-growing anaerobic bacteria. The genus includes both pathogenic and non-pathogenic species, and these variously colonise the large intestines of different species of birds and animals, including humans. Scientific understanding of the physiology and molecular biology of Brachyspira spp. remains very limited compared with that of other pathogenic spirochetes, and there are few descriptions of successful genetic manipulations undertaken to investigate gene function. An important boost to knowledge occurred in 2009 when, for the first time, the whole genome sequence of a Brachyspira strain (Brachyspira hyodysenteriae strain WA1) was obtained. The genomics analysis provided a significant increase in knowledge: for example, a previously unknown ~36 Kb plasmid was discovered and metabolic pathways were constructed. The study also revealed likely acquisition of genes involved in transport and central metabolic functions from other enteric bacterial species. Four subsequent publications have provided a similarly detailed analysis of other Brachyspira genomes, but of these only two included more than one strain of a species (20 strains of B. hyodysenteriae in one and three strains of B. pilosicoli in the other). Since then, more Brachyspira genomes have been made publicly available, with the sequences of at least one representative of each of the nine officially recognised species deposited at public genome repositories. All species have a single circular chromosome varying in size from ~2.5 to 3.3 Mb, with a C + G content of around 27%. In this chapter, we summarise the current knowledge and present a preliminary comparative genomic analysis conducted on 56 strains covering the official Brachyspira species. Besides providing detailed genetic maps of the bacteria, this analysis has revealed gene island rearrangements, putative phenotypes (including antimicrobial drug resistance) and genetic mutation mechanisms that enable brachyspires to evolve and respond to stress. The application of Next-Generation Sequencing (NGS) to generate genomic data from many more Brachyspira species and strains increasing will improve our understanding of these enigmatic spirochetes.

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

The 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 HIGHLIGHTS Laying 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.

Antibiotic treatment triggers gut dysbiosis and modulates metabolism in a chicken model of gastro-intestinal infection.

BACKGROUND: Infection of the digestive track by gastro-intestinal pathogens results in the development of symptoms ranging from mild diarrhea to more severe clinical signs such as dysentery, severe dehydration and potentially death. Although, antibiotics are efficient to tackle infections, they also trigger dysbiosis that has been suggested to result in variation in weight gain in animal production systems. RESULTS: Here is the first study demonstrating the metabolic impact of infection by a gastro-intestinal pathogen (Brachyspira pilosicoli) and its resolution by antibiotic treatment (tiamulin) on the host (chicken) systemic metabolism and gut microbiota composition using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy and 16S rDNA next generation sequencing (NGS). Clear systemic metabolic markers of infections such as glycerol and betaine were identified. Weight loss in untreated animals was in part explained by the observation of a modification of systemic host energy metabolism characterized by the utilization of glycerol as a glucose precursor. However, antibiotic treatment triggered an increased VLDL/HDL ratio in plasma that may contribute to reducing weight loss observed in treated birds. All metabolic responses co-occurred with significant shift of the microbiota upon infection or antibiotic treatment. CONCLUSION: This study indicates that infection and antibiotic treatment trigger dysbiosis that may impact host systemic energy metabolism and cause phenotypic and health modifications.

Brachyspira catarrhinii sp. nov., an anaerobic intestinal spirochaete isolated from vervet monkeys may have been misidentified as Brachyspira aalborgi in previous studies.

To date nine species of anaerobic intestinal spirochaetes have been validly assigned to the genus Brachyspira. These include both pathogenic and non-pathogenic species. In the current study a genomic analysis of a novel spirochaete isolate was undertaken to determine whether it is a distinct species that previously has been misidentified as Brachyspira aalborgi. The genome of spirochaete strain Z12 isolated from the faeces of a vervet monkey was sequenced and compared to the genomes of the type strains of the nine assigned Brachyspira species. Genome to Genome Distance (GGD) values and Average Nucleotide Identity (ANI) values were determined. Single nucleotide polymorphisms (SNP) were used to create a phylogenetic tree to assess relatedness. The 16S rRNA gene sequences of the strains were aligned and the similarity amongst the Brachyspira species was recorded. Multilocus sequence typing (MLST) using five loci was conducted on Z12 and results compared with those for other Brachyspira isolates. Assembly of the Z12 sequences revealed a 2,629,108 bp genome with an average G + C content of 31.3%. The GGD, ANI, 16S rRNA gene sequence comparisons and the MLST results all indicated that Z12 represents a distinct species within the genus Brachyspira, with its nearest neighbour being B. aalborgi. Spirochaete strain Z12T was assigned as the type strain of a new species, Brachyspira catarrhinii sp. nov. The diagnostic PCR currently in use to detect B. aalborgi cross-reacts with Z12, but RFLP analysis of PCR product can be used to distinguish the two species. Previous reports of non-human primates being colonised by B. aalborgi based on PCR results may have been incorrect. The development of an improved diagnostic method will allow future studies on the distribution and possible clinical significance of these two anaerobic spirochaete species.

High prevalence of Brachyspira spp. in layers kept in alternative husbandry systems associated with frequent species variations from end of rearing to slaughter.

A longitudinal survey was conducted to investigate the presence of Brachyspira species in layer flocks. A total of 66 layer flocks kept in alternative husbandry systems were sampled at three time points: end of rearing, at peak of lay and at end of lay. Content from caecal samples of freshly killed birds was cultured at each sampling time point and processed for further investigations. Gross pathological lesions in caeca were recorded during post mortem investigation. Spirochaetes were isolated from 50 flocks: three flocks were positive at all three sampling points, 25 flocks at two and 22 flocks at one sampling point, respectively. The presence of Brachyspira spp. could not be related to specific gross pathological caecal lesions or antibiotic treatments. The number of positive flocks increased with the age of birds. Furthermore, organic flocks were more often positive than flocks from barn systems. In total 80 spirochaetal cultures were obtained. B. intermedia (43.8%) was the most common species, followed by B. pulli (13.8%) and B. pilosicoli (12.5%). Brachyspira murdochii and B. innocens were found in 5.0% and 2.5%, respectively, whereas 11.3% of Brachyspira isolates could not be identified to species level. In 11.3% of the samples mixed infections were detected. Finally, the longitudinal survey revealed for the first time a possible shift in the Brachyspira species in a substantial number (32%) of layer flocks during their lifetime.

A novel multiplex qPCR targeting 23S rDNA for diagnosis of swine dysentery and porcine intestinal spirochaetosis.

BACKGROUND: A multiplex qPCR targeting a 128 bp region on the 23S rDNA gene was developed for detection of Brachyspira (B.) hyodysenteriae and B. pilosicoli, the agents of swine dysentery (SD) and porcine intestinal spirochaetosis (PIS), together with a triplet of apathogenic Brachyspira spp. (B. innocens, B. intermedia, B. murdochii) in porcine feces. The multiplex qPCR was evaluated against a duplex PCR (La et al., J Clin Microbiol 41:3372-5, 2003). RESULTS: Using DNA extracted from fecal culture, the multiplex qPCR showed excellent agreement with the duplex PCR (κ = 0.943 and 0.933). In addition, thanks to the three probes whereof one detecting the apathogenic Brachyspria spp., a more diversified overview of the brachyspiral flora in porcine fecal samples can be delivered as a part of the routine diagnostic. The multiplex qPCR with a limit of detection of 5-10 genomic equivalents (GE) per reaction (6 × 102 GE per gram) allows reliable detection of Brachyspira species directly from fecal swab DNA. In line with this, analysis of 202 fecal swabs in comparison with culture-based qPCR showed a high agreement for the causative agents of SD (B.hyodysenteriae: κ = 0.853, sensitivity 87% specificity 98%). CONCLUSION: The novel multiplex qPCR is robust and has a high analytical sensitivity and is therefore suitable for high-throughput screening of porcine fecal swabs for the causative agents of SD. This assay can therefore be used for the direct proof of the pathogenic B. spp. in fecal swabs within the scope of a monitoring program.

An optimized swine dysentery murine model to characterize shedding and clinical disease associated with "Brachyspira hampsonii" infection.

BACKGROUND: The development of a mouse model as an in vivo pathogenicity screening tool for Brachyspira spp. has advanced the study of these economically important pathogens in recent years. However, none of the murine models published to date have been used to characterize the clinical signs of disease in mice, instead focusing on pathology following oral inoculation with various Brachyspira spp. The experiments described herein explore modifications of published models to characterize faecal consistency, faecal shedding and pathology in mice challenged with "Brachyspira hampsonii" clade II (Bhamp). METHODS AND RESULTS: In Experiment 1, 24 CF-1 mice were randomly allocated to one of three inoculation groups: sham (Ctrl), Bhamp, or B. hyodysenteriae (Bhyo; positive control). Half of each group was fed normal mouse chow (RMH) while the other received a low-zinc diet (TD85420). In Experiment 2, eight CF-1 mice and nine C3H/HeN mice were divided into Ctrl or Bhamp inoculation groups, and all fed TD85420. In Experiment 1, mice fed TD85420 demonstrated more severe mucoid faeces (P = 0.001; Kruskal Wallis) and faecal shedding for a significantly greater number of days (P = 0.005; Kruskal Wallis). Mean faecal scores of Bhamp inoculated mice trended higher than Ctrl (P = 0.06; Wilcoxon rank-sum) as did those of Bhyo mice (P = 0.0; Wilcoxon rank-sum). In Experiment 2, mean faecal scores of inoculated CF-1 mice were significantly greater than in C3H mice (P = 0.049; Kruskal Wallis) but no group differences in faecal shedding were observed. In both experiments, mice clustered based on the severity of colonic and caecal histopathology but high lesion scores were not always concurrent with high fecal scores. CONCLUSION: In our laboratory, CF-1 mice and the lower-zinc TD85420 diet provide a superior murine challenge model of "Brachyspira hampsonii" clade II.