Brachyspira Hampsonii Research Articles

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. Thepresent 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.

Chaos in the Colon: Unlocking the Puzzle of DRA (SLC26A3) Downregulation with IL-1α and p38 MAPK Amidst Brachyspira Turmoil

This study examines the impact of Brachyspira hyodysenteriae and Brachyspira hampsonii infection on chloride (36Cl−) transport within the porcine colon. Utilizing 3H-mannitol and 36Cl−, we evaluated the bidirectional flux across colonic tissues from both healthy and infected pigs. We observed no change in 3H-mannitol flux, indicating unaffected paracellular movement. However, we observed a significant decrease in mucosal-serosal 36Cl− flux (Jm-s) in the apical segment of the spiral colon of infected pigs, suggesting a compromised chloride absorption due to the downregulation of DRA (SLC26A3), confirmed by reduced mRNA and protein levels. To dissect the molecular dynamics in vitro, we exposed polarized Caco-2 cells to B. hampsonii lysate. This model recapitulated the in vivo responses, including the downregulation of DRA and the concomitant IL-1α upregulation. This in vitro model demonstrated that B. hampsonii lysate upregulates IL-1α expression through p38 MAPK phosphorylation. More importantly, in vitro studies with B. hampsonii lysate, recombinant IL-1α, SB 203580 (p38 MAPK inhibitor), and blocking IL-1α signalling with IL-1 receptor antagonist (IL-1RA) revealed that IL-1α has a dose-dependent negative effect on DRA expression and a positive effect on IL-1α expression. Furthermore, inhibiting IL-1α expression with SB 203580 and blocking IL-1α signalling with IL-1RA not only prevented this downregulation of DRA but also promoted its upregulation, highlighting the regulatory role of IL-1α in chloride absorption. In summary, this study presents an in-depth understanding by which Brachyspira infections can impede chloride absorption in the colon and offers potential intervention targets for conditions involving impaired DRA-mediated Cl− transport. This research was supported by Alberta Livestock Meat Association (ALMA 2013R054R) and Natural Sciences and Engineering Research Council of Canada Discovery Grant 02743-2021 (to M. E. Loewen). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

T-independent B-cell effect of agents associated with swine grower-finisher diarrhea.

Swine dysentery, spirochetal colitis, and salmonellosis are production-limiting enteric diseases of global importance to the swine industry. Despite decades of efforts, mitigation of these diseases still relies on antibiotic therapy. A common knowledge gap among the 3 agents is the early B-cell response to infection in pigs. Thus, this study aimed to characterize the porcine B-cell response to Brachyspira hyodysenteriae, Brachyspira hampsonii (virulent and avirulent strains), Brachyspira pilosicoli, and Salmonella Typhimurium, the agents of the syndromes mentioned above. Immortalized porcine B-cell line derived from a crossbred pig with lymphoma were co-incubated for 8h with each pathogen, as well as E. coli lipopolysaccharide (LPS) and a sham-inoculum (n = 3/treatment). B-cell viability following treatments was evaluated using trypan blue, and the expression levels of B-cell activation-related genes was profiled using reverse transcription quantitative PCR. Only S. Typhimurium and LPS led to increased B-cell mortality. B. pilosicoli downregulated B-lymphocyte antigen (CD19), spleen associated tyrosine Kinase (syk), tyrosine-protein kinase (lyn), and Tumour Necrosis Factor alpha (TNF-α), and elicited no change in immunoglobulin-associated beta (CD79b) and swine leukocyte antigen class II (SLA-DRA) expression levels, when compared to the sham-inoculated group. In contrast, all other treatments significantly upregulated CD79b and stimulated responses in other B-cell downstream genes. These findings suggest that B. pilosicoli does not elicit an immediate T-independent B-cell response, nor does it trigger antigen-presenting mechanisms. All other agents activated at least one trigger within the T-independent pathways, as well as peptide antigen presenting mechanisms. Future research is warranted to verify these findings in vivo.

Antimicrobial susceptibility of U.S. porcine Brachyspira isolates and genetic diversity of B. hyodysenteriae by multilocus sequence typing.

Swine dysentery, caused by Brachyspira hyodysenteriae and the newly recognized Brachyspira hampsonii in grower-finisher pigs, is a substantial economic burden in many swine-rearing countries. Antimicrobial therapy is the only commercially available measure to control and prevent Brachyspira-related colitis. However, data on antimicrobial susceptibility trends and genetic diversity of Brachyspira species from North America is limited. We evaluated the antimicrobial susceptibility profiles of U.S. Brachyspira isolates recovered between 2013 and 2022 to tiamulin, tylvalosin, lincomycin, doxycycline, bacitracin, and tylosin. In addition, we performed multilocus sequence typing (MLST) on 64 B. hyodysenteriae isolates. Overall, no distinct alterations in the susceptibility patterns over time were observed among Brachyspira species. However, resistance to the commonly used antimicrobials was seen sporadically with a higher resistance frequency to tylosin compared to other tested drugs. B. hampsonii was more susceptible to the tested drugs than B. hyodysenteriae and B. pilosicoli. MLST revealed 16 different sequence types (STs) among the 64 B. hyodysenteriae isolates tested, of which 5 STs were previously known, whereas 11 were novel. Most isolates belonged to the known STs: ST93 (n = 32) and ST107 (n = 13). Our findings indicate an overall low prevalence of resistance to clinically important antimicrobials other than tylosin and bacitracin, and high genetic diversity among the clinical Brachyspira isolates from pigs in the United States during the past decade. Further molecular, epidemiologic, and surveillance studies are needed to better understand the infection dynamics of Brachyspira on swine farms and to help develop effective control measures.

Experimental natural transmission (seeder pig) models for reproduction of swine dysentery.

Swine dysentery is causally associated with Brachyspira hampsonii and B. hyodysenteriae infection. Given the importance of transmission models in understanding re-emergent diseases and developing control strategies such as vaccines, the objective of this experiment was to evaluate two experimental natural transmission (seeder pig) models in grower pigs, each with 24 animals. Seeder pigs were intragastrically inoculated using broth cultures of either B. hampsonii strain 30446 (genomovar II) or B. hyodysenteriae strain G44. In trial 1, three seeder pigs were placed into two pens containing nine susceptible contact pigs creating a 1:3 seeder:contact ratio. This was sufficient to achieve natural B. hampsonii infection of 13/18 (72%) contact pigs, however, the incidence of mucoid or mucohemorrhagic diarrhea (MMHD) in contact pigs differed significantly between pens (4/9 versus 9/9; P = 0.03). In trial 2, eight seeder pigs inoculated intragastrically with B. hampsonii did not develop MMHD but when re-inoculated with B. hyodysenteriae 14 days later, all developed mucohemorrhagic diarrhea within 13 days of re-inoculation. Two seeder pigs were placed into each of 4 contact pens each containing 4 pigs. This 1:2 seeder:contact ratio resulted in natural infection of 14/16 (87%) contact pigs with incubation period ranging from 9–15 days. There were no significant differences among pens in incubation period, duration, clinical period or severity of diarrhea. These trials demonstrated that a 1:2 seeder:contact ratio with groups of six grower pigs per pen sustained natural transmission of B. hyodysenteriae G44 with greater consistency in the incidence of MMHD among pens compared to a B. hampsonii 30446 transmission model using 1:3 seeder:contact ratio in pens of 12. Understanding why B. hampsonii intragastric inoculation failed in one experiment warrants additional research.

Metagenomics of the midgut microbiome of Rhipicephalus microplus from China

BackgroundTicks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods.MethodsRhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species’ sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison.ResultsThe dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcuspneumonia infection, human granulocytic anaplasmosis, Shigellasonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer.ConclusionsOur study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick’s midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases.Graphical

Mutational analysis of TlyA from Brachyspira hampsonii reveals two key residues conserved in pathogenic bacteria responsible for oligomerization and hemolytic activity

BackgroundTlyA proteins are expressed in a variety of pathogenic bacteria and possess dual hemolytic and ribosomal RNA methyltransferase functions. While the mechanism of TlyA mediated rRNA methylation is well understood, relatively little is known about the mechanism of TlyA induced hemolysis. MethodsTlyA protein from the pig pathogen Brachyspira hampsonii was heterologously expressed and purified from an E. coli host. Hemolytic activity and rRNA methylation were assessed in vitro. Site-directed mutagenesis was used to mutate amino acids believed to be involved in TlyA mediated hemolysis. ResultsPurified TlyA-His protein exhibited both hemolytic and rRNA methyltransferase activities in vitro, with partial inhibition of hemolysis observed under reducing conditions. Mutation of cysteine 80 to alanine impaired hemolytic activity. A C27A/C93A mutant was capable of dimerizing under non-reducing conditions, indicating that a C80-C80 disulfide bond is involved in TlyA oligomerization. A mutation conserved in several avirulent Brachyspira species (S9K) completely abolished hemolytic activity of TlyA. This loss of activity was attributed to impaired oligomerization in the S9K mutant, as assessed by ITC and size-exclusion chromatography experiments. ConclusionsOligomeric assembly and hemolytic activity of TlyA from Brachyspira hampsonii is dependent on the formation of an intermolecular C80-C80 disulfide bond and noncovalent interactions involving serine 9. The conservation of these amino acids in TlyA proteins from pathogenic bacteria suggests a correlation between tlyA gene mutations and bacterial virulence. General significanceOur results further elucidate the mechanisms underlying TlyA mediated hemolysis and provide evidence of a conserved mechanism of oligomerization for TlyA family proteins.

Retrospective analysis of necrotizing typhlitis cases associated with Brachyspira spp. in British rheas

ABSTRACT This paper describes a retrospective analysis of necrotizing typhlitis in common rheas (Rhea americana) diagnosed in the United Kingdom by the Animal & Plant Health Agency (APHA). From January 2008 to January 2020, seven cases of spirochaetal typhlitis associated with Brachyspira spp. were identified using the Veterinary Investigation Diagnosis Analysis database. Gross examination was combined with selective anaerobic culture, polymerase chain reaction, and histopathology to diagnose typhlitis associated with spirochaetal infection. Whole-genome sequencing was subsequently utilized on archived isolates from six of the seven submissions, overcoming issues with traditional testing methods and yielded gains in the identification of Brachyspira to species level. Brachyspira hyodysenteriae, an organism traditionally associated with typhlitis in rheas, was isolated in three sequenced submissions. One of these also demonstrated co-infection with Brachyspira intermedia. Brachyspira suanatina, Brachyspira hampsonii, and Brachyspira alvinipulli were identified by sequencing as single infections in the remaining three animals. This report demonstrates the ability of Brachyspira species other than B. hyodysenteriae to colonize the caeca of rheas presenting with typhlitis. Additionally, the B. alvinipulli isolate harboured a tva(A) gene, indicating higher potential pleuromutilin resistance, which has not previously been described in this Brachyspira species. This study discusses the epidemiology of examined cases and examines the potential role other species may play in these outbreaks.

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.

Subclinical colitis associated with moderately hemolytic Brachyspira strains

Objective: Microbiological and virulence characterization of 2 moderately hemolytic Brachyspira strains. Materials and methods: Clinical isolates were obtained from diarrheic (3603-F2) and healthy (G79) pigs. Phenotypic characterization included assessment of hemolytic activity on blood agar and biochemical profiling. Genotyping was performed by sequencing the nicotinamide adenine dinucleotide oxidase (nox) gene, whole genome sequencing, and comparison to relevant Brachyspira. Pig inoculation included 4 treatment groups in 2 challenge experiments: negative control (sterile broth media; n = 12), positive control (Brachyspira hampsonii genomovar 2 strain 30446; n = 18), and 3603-F2 (n = 12) or G79 (n = 12). Fecal scoring and rectal swabbing for culture were performed daily. Animals were euthanized following onset of mucohemorrhagic diarrhea or between 21 and 28 days post inoculation (dpi). Gross and microscopic pathology were assessed. Terminal colon samples were used to characterize post-infection mucosal ion secretion. Results: Both strains were moderately hemolytic. Whole genome and nox sequencing identified 3603-F2 as Brachyspira murdochii and G79 as a novel strain. Both challenge trials revealed intestinal colonization, but no mucohemorrhagic diarrhea. Sporadic watery diarrhea was induced by 3603-F2 associated with a pattern of microscopic lesions similar to pigs with swine dysentery (positive controls). No diarrhea was observed in G79 inoculated pigs, but microscopic lesions were more severe than in controls. Both strains induced greater colonic anion secretory potential than negative controls 21 dpi. Implications: Allegedly avirulent Brachyspira species most closely related to B murdochii can be associated with subclinical colitis and may be a concern for grow-finish pigs.

Decreased electrogenic anionic secretory response in the porcine colon following in vivo challenge with Brachyspira spp. supports an altered mucin environment.

Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( Isc) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable Isc. Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization. NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host's cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

Retrospective detection of Brachyspira hampsonii in archived colitis cases from western Canadian swine.

Mucohaemorrhagic diarrhea associated with Brachyspira hampsonii infection has emerged as a production-limiting disease in western Canada. This pathogen was first described in North America in 2010, and reports of its detection occurred concurrently in western Canada and the United States. Since that time, Brachyspira hampsonii has been detected in Europe, both in pigs and in waterfowl. The origin of B.hampsonii and the timing and reasons for its emergence are unknown. We conducted a retrospective study of historic, archived cases of porcine colitis to determine when B.hampsonii was first evident in western Canada. A total of 206 samples from 114 cases submitted from 57 different farms or productions systems in 1984 and 1999-2009 were screened using real-time PCR assays targeting B.hampsonii genomovars I and II, and Brachyspira hyodysenteriae (the traditional agent of swine dysentery). In most cases, positive real-time PCR results were confirmed by amplification and sequencing of additional gene targets. A total of 9, 7 and 5 samples tested positive for B.hampsonii (I), B.hampsonii (II) or B.hyodysenteriae respectively. The results of this study push the date of first appearance of B.hampsonii in pigs in western Canada back to 2002 (B.hampsonii (I)) and 2006 (B.hampsonii (II)), which is up to 7years before the new species were first identified in fresh samples.

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.

Infection of porcine colon explants with "Brachyspira hampsonii" leads to increased epithelial necrosis and catarrhal exudate.

Mucohemorrhagic diarrhea in pigs caused by Brachyspira spp. has a global distribution, and an economic impact on affected farms due to poor performance of animals. Demonstrations that "Brachyspira hampsonii" is pathogenic have been achieved using in vivo animal models, but a critical knowledge gap exists regarding the pathogenic mechanisms employed by Brachyspira. Here, we used in vitro organ culture of porcine colon to investigate interactions between "B. hampsonii" and explants during the first 12 h of contact. Explants were either inoculated with "B. hampsonii" or sterile culture broth. Responses to infection were evaluated by optical microscopy and quantitative PCR. Significantly greater numbers of necrotic crypt cells and thicker catarrhal exudate were observed on infected explants compared to controls. Spirochaetes were observed in the mucus layer, in contact with necrotic exfoliated cells, in crypts and the lamina propria. Statistical differences were observed in mRNA levels between inoculated and control explants for IL-1α, TNF-α and ZO-1 using a Bayesian analysis, but not observed using the ΔΔCq method. These results provide a demonstration of a porcine colon explant model for investigating interactions of Brachyspira with its host and show that initial effects on the host are observed within the first 12 h of contact.

Characterization of “Brachyspira hampsonii” clades I and II isolated from commercial swine in Western Canada

A novel Brachyspira emerged in 2009 and has since become a production-limiting pathogen of pigs in North America. The name “ Brachyspira hampsonii” has been proposed for this novel taxon. “ Brachyspira hampsonii” is divided into two phylogenetically distinct clades based on the sequence of the NADH-oxidase ( nox) gene, although the clinical disease associated with clades I and II is indistinguishable and phenotypic characteristics that discriminate the clades have not been determined. The objectives of the current study were to enhance the description of the provisional species “ B. hampsonii” with biochemical profiles and morphometric data from isolates affecting Canadian swine and to investigate potentially diagnostically informative characteristics for this emerging pathogen. Biochemical profiles of isolates from different commercial swine barns in Western Canada showed that biochemical profiles were insufficient to distinguish “ B. hampsonii” clades I and II from each other or from other pathogenic Brachyspira. Hippurate hydrolysis, previously reported as uniformly negative in “ B. hampsonii,” was variable among Canadian isolates. Spirochete dimensions and flagella numbers for “ B. hampsonii” overlapped with other Brachyspira species. Taken together, these results indicate that nox gene sequencing remains a preferred method for identification and discrimination of “ B. hampsonii” from other pig-associated Brachyspira spp.

Characterization and Recognition of Brachyspira hampsonii sp. nov., a Novel Intestinal Spirochete That Is Pathogenic to Pigs.

Swine dysentery (SD) is a mucohemorrhagic colitis of swine classically caused by infection with the intestinal spirochete Brachyspira hyodysenteriae Since around 2007, cases of SD have occurred in North America associated with a different strongly beta-hemolytic spirochete that has been molecularly and phenotypically characterized and provisionally named "Brachyspira hampsonii." Despite increasing international interest, B. hampsonii is currently not recognized as a valid species. To support its recognition, we sequenced the genomes of strains NSH-16T, NSH-24, and P280/1, representing B. hampsonii genetic groups I, II, and III, respectively, and compared them with genomes of other valid Brachyspira species. The draft genome of strain NSH-16T has a DNA G+C content of 27.4% and an approximate size of 3.2 Mb. Genomic indices, including digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI), clearly differentiated B. hampsonii from other recognized Brachyspira species. Although discriminated genotypically, the three genetic groups are phenotypically similar. By electron microscopy, cells of different strains of B. hampsonii measure 5 to 10 μm by 0.28 to 0.34 μm, with one or two flat curves, and have 10 to 14 periplasmic flagella inserted at each cell end. Using a comprehensive evaluation of genotypic (gene comparisons and multilocus sequence typing and analysis), genomic (dDDH, ANI, and AAI) and phenotypic (hemolysis, biochemical profiles, protein spectra, antibiogram, and pathogenicity) properties, we classify Brachyspira hampsonii sp. nov. as a unique species with genetically diverse yet phenotypically similar genomovars (I, II, and III). We designate the type strain NSH-16 (= ATCC BAA-2463 = NCTC 13792).

First screening for Brachyspira hampsonii in Swiss pigs applying a new high resolution melting assay

A new High Resolution Melting (HRM) assay was developed for the rapid detection of Brachyspira (B.) hampsonii. B. hampsonii occurs in different European countries, however, until today it has not been encountered in Switzerland.Four B. hampsonii reference strains were used to develop the HRM assay: B. hampsonii clade I ATCC BAA2463 and clade II ATCC BAA2464 strain, as well as two isolated strains P280/1 from the UK and the German isolate 5369-1x/12. A conserved region of the nox gene was used to design B. hampsonii-specific primers. The HRM melting curves for the four reference strains showed reproducible difference graphs with distinct differences between the four strains based on a slight variation between the four amplicon sequences. In addition, DNA from 22 B. hampsonii strains representing four genetic B. hampsonii groups was used to validate the method. Melting temperatures in the interval between 73.1 and 74°C were obtained for all B. hampsonii strains and allow differentiating B. hampsonii from other Brachyspira species.In total 897 Swiss porcine fecal Brachyspira isolates, cultured between 2009 and 2015, were analysed by the HRM protocol. B. hampsonii was not detected among these Swiss Brachyspira isolates. In conclusion, the rapid and low-cost HRM approach allows a sensitive and specific identification of B. hampsonii.

Antimicrobial Susceptibility Patterns of Brachyspira Species Isolated from Swine Herds in the United States

Outbreaks of swine dysentery, caused by Brachyspira hyodysenteriae and the recently discovered "Brachyspira hampsonii," have reoccurred in North American swine herds since the late 2000s. Additionally, multiple Brachyspira species have been increasingly isolated by North American diagnostic laboratories. In Europe, the reliance on antimicrobial therapy for control of swine dysentery has been followed by reports of antimicrobial resistance over time. The objectives of our study were to determine the antimicrobial susceptibility trends of four Brachyspira species originating from U.S. swine herds and to investigate their associations with the bacterial species, genotypes, and epidemiological origins of the isolates. We evaluated the susceptibility of B. hyodysenteriae, B. hampsonii, Brachyspira pilosicoli, and Brachyspira murdochii to tiamulin, valnemulin, doxycycline, lincomycin, and tylosin by broth microdilution and that to carbadox by agar dilution. In general, Brachyspira species showed high susceptibility to tiamulin, valnemulin, and carbadox, heterogeneous susceptibility to doxycycline, and low susceptibility to lincomycin and tylosin. A trend of decreasing antimicrobial susceptibility by species was observed (B. hampsonii > B. hyodysenteriae > B. murdochii > B. pilosicoli). In general, Brachyspira isolates from the United States were more susceptible to these antimicrobials than were isolates from other countries. Decreased antimicrobial susceptibility was associated with the genotype, stage of production, and production system from which the isolate originated, which highlights the roles of biosecurity and husbandry in disease prevention and control. Finally, this study also highlights the urgent need for Clinical and Laboratory Standards Institute-approved clinical breakpoints for Brachyspira species, to facilitate informed therapeutic and control strategies.

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

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

Characterization of Heterologously Expressed Putative Cytolysins from “Brachyspira hampsonii “

Cytolysins produced by Brachyspira hyodysenteriae and “Brachyspira hampsonii” strain 30446 are believed to be important virulence factors in the pathology of swine dysentery. To date four putative cytolysin genes termed tlyA, tlyB, tlyC, and hlyA have been identified, all of which are present in both Brachyspira hyodysenteriae and “Brachyspira hampsonii”. While the contributions of tlyB, tlyC, and hlyA to the pathogenesis of swine dysentery are not yet known, tlyA knockout strains of Brachyspira hyodysenteriae have shown decreased virulence in mouse models. In addition, homologues of tlyA are believed to be important virulence factors for human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, functioning as both cytolysins and contributing to antibiotic resistance through ribosomal RNA methylation. While active hlyA protein has been purified from Brachyspira hyodysenteriae broth cultures, studies of tlyA, tlyB, and tlyC to date have focused on their activity when introduced into Escherichia coli via plasmid. These results were cast into doubt several years later upon the discovery that many lab strains of Escherichia coli exhibit a hemolytic phenotype when foreign genes were introduced. To examine the effects of these putative cytolysins the tlyA and tlyC genes were amplified from “Brachyspira hampsonii” genomic DNA and ligated into expression vectors. The resulting protein products were overexpressed in Escherichia coli and purified by Ni2+ and amylose affinity chromatography. Cytolytic activity of purified recombinant tlyA and tlyC on a sheep erythrocyte suspension was examined, and no cytolysis greater than a phosphate buffered saline control was observed from tlyA at concentrations ranging from 0 to 88 μg/mL or from tlyC at concentrations ranging from 0–34 μg/mL. At the time of writing, attempts are being made to express and purify the α‐toxin from Staphylococcus aureus utilizing the same methodology in order to rule out inactivation of tlyA and tlyC by the purification process. While it is also possible that higher concentrations of tlyA and tlyC are required for hemolysis or that full activity of these proteins require unidentified cofactors or post‐translational modifications not possible in our current Escherichia coli based expression system, the possibility that tlyA and tlyC have been misannotated as cytolysins cannot be ruled out.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) 371364 /2010 to MEL & Alberta Livestock and Meat Association project 2013R054R to JCH, MEL & JEH