Isolation and characterization of bacteria residing in the oral, gut, and fecal samples of different pheasant species / Isolamento e caracterização de bactérias residentes nas amostras orais, intestinais e fecais de diferentes espécies de faisão

There is a paucity of research conducted on microbial prevalence in pheasants. The microbiota of captive birds has zoonotic significance and must be characterize. Present study is therefore planned to assess the microbiota from oral, fecal and gut content of captive avian species. It will be helpful in characterization of harmful microbes. Different samples taken from oral, gut and feces of ring-necked pheasants (Phasianus colchicus), green pheasants (Phasianus versicolor), golden pheasant (Chrysolophus pictus) and silver pheasant (Lophura nycthemera). Samples were collected, diluted, and inoculated onto different agar plates (MacConkey, SS agar, MSA and nutrient agar) for cultivation of bacterial species. Colonies of E.coli, Staphylococcus spp. Brachyspira spp. and Campylobacter spp were observed based on colony morphology. Colony forming unit showed E. coli as frequently found bacteria in fecal, oral and gut contents of all the above pheasants. The overall significance difference was found among bacterial species of golden pheasants, green pheasant, ring-necked pheasant, and silver pheasants. It was concluded that E.coli is predominant isolated from heathy pheasants followed by Campylobacter, Staphylococcus and Brachyspira.

Há uma escassez de pesquisas realizadas sobre a prevalência microbiana em faisões. A microbiota de aves em cativeiro tem significado zoonótico e deve ser caracterizada. O presente estudo está, portanto, planejado para avaliar a microbiota do conteúdo oral, fecal e intestinal de espécies aviárias em cativeiro. Será útil na caracterização de micróbios nocivos. Diferentes amostras retiradas da boca, intestino e fezes de faisões de pescoço redondo (Phasianus colchicus), faisões verdes (Phasianus versicolor), faisões dourados (Chrysolophus pictus) e faisão prateado (Lophura nycthemera). As amostras foram coletadas, diluídas e inoculadas em diferentes placas de ágar (MacConkey, ágar SS, MSA e ágar nutriente) para o cultivo de espécies bacterianas. Colônias de E. coli, Staphylococcus spp., Brachyspira spp. e Campylobacter spp foram observados com base na morfologia da colônia. A unidade formadora de colônia mostrou E. coli como bactéria frequentemente encontrada no conteúdo fecal, oral e intestinal de todos os faisões acima. A diferença de significância geral foi encontrada entre as espécies bacterianas de faisões dourados, faisões verdes, faisões de pescoço anelado e faisões prateados. Verificou-se que a E.coli é predominantemente isolada de faisões saudáveis, seguida por Campylobacter, Staphylococcus e Brachyspira.

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.

Una causa infrecuente de diarrea crónica / A rare cause of chronic diarrea

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Severe Colitis with Portal Venous Gas Caused by Brachyspira pilosicoli Infection.

We herein report a case of Brachyspira pilosicoli-caused severe colitis presenting with portal venous gas. A 75-year-old man was admitted because of a fever, severe abdominal pain and bloody diarrhea. He was negative for anti-HIV antibodies. He had been in close contact with a dog earlier. Abdominal computed tomography detected severe wall-thickening and fat-stranding of the entire colon accompanied by portal venous gas. A smear examination of his stool showed many Gram-negative spiral rods, suggesting intestinal spirochetosis. A polymerase chain reaction assay using stool samples detected an amplified band specific for B. pilosicoli. He responded well to antimicrobial agents including metronidazole.

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.

Diagnosis of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in hens and laying hens in the western region of Paraná through bacterial isolation and identification in qPCR / Diagnóstico de Brachyspira pilosicoli, Brachyspira hyodysenteriae e Brachyspira intermedia em aves de postura e matrizes de corte na região oeste do Paraná através do isolamento bacteriano e identificação na qPCR

Bacteria of the genus Brachyspira can cause enteric diseases in poultry causing a decrease in productivity. The occurrence of this disease in chickens has already been verified in countries such as Australia, Italy, and the United States, but in Brazil, until now, epidemiological studies about Brachyspira sp. frequency were only carried out on pig farms. The objective of this study was to evaluate the presence of bacteria of the genus Brachyspira sp. through isolation and confirmation of the species Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia using the qPCR technique. Samples from 110 hens aged from 35 to 82 weeks were collected, 40 were from commercial egg farms and 70 were from laying hens matrices. For the first evaluation, bacterial isolation was performed from the feces. Positive samples were submitted to qPCR to identify the three species proposed. Cecum fragments of the birds were collected and fixed in formaldehyde for histological evaluation and counting of goblet cells. Of the 110 samples, 48 characteristic isolates of Brachyspira (43.6%) were obtained and of these in qPCR 13 identified as B. hyodysenteriae (11.8%) and 5 all from the same farm as Brachyspira intermedia (4.5%), 2 samples were positive for both agents (1.8%) and 28 were not characterized by qPCR (25.5%). None histopathological lesions were observed in the chicken cecum and no significant statistical difference was noticed in the count of goblet cells of the positive hens. It can be evidenced by the occurrence of Brachyspira sp. in laying farms and hens in Brazil, with special relevance to Brachyspira intermedia that can be potentially pathogenic for these animals.(AU)

Bactérias do gênero Brachyspira podem ocasionar enfermidades entéricas em aves acarretando a queda de produtividade. A ocorrência desta enfermidade em galinhas já foi verificada em países como a Austrália, Itália e Estados Unidos, porém no Brasil, até o momento, trabalhos epidemiológicos sobre a frequencia de Brachyspira sp. só foram realizados em granjas de suínos. O objetivo deste estudo foi avaliar a presença de bactérias do gênero Brachyspira sp. através do isolamento e confirmação das espécies Brachyspira pilosicoli, Brachyspira hyodysenteriae e Brachyspira intermedia utilizando a técnica de qPCR. Foram coletadas amostras de 110 aves com idade entre 35 e 82 semanas, sendo 40 de granjas de postura comercial e 70 de granjas de matrizes de corte. Para avaliação primeiramente procedeu-se o isolamento bacteriano a partir das fezes. As amostras positivas foram submetidas a qPCR para identificação das três espécies propostas. Fragmentos de ceco das aves foram coletados e fixados em formol para avaliação histológica e contagem de células caliciformes. Das 110 amostras foram obtidos 48 isolamentos característicos de Brachyspira (43,6%) e destes na qPCR 13 identificadas como B. hyodysenteriae (11,8%) e 5 sendo todas da mesma granja (4,5%) como B. intermedia, 2 amostras foram positivas para ambos os agentes (1,8%) e 28 não foram caracterizadas através da qPCR (25,5%). Não foram observadas alterações histopatológicas no ceco e diferença estatística significativa na contagem de células caliciformes das aves positivas. Conclui-se que a Brachyspira sp. é frequente em granjas de poedeiras e matrizes de corte no Brasil, com especial relevância para a B. intermedia que possui potência patogênico para estas aves.(AU)

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.

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 7 years before the new species were first identified in fresh samples.

Simultaneous isolation of two species, Brachyspira pilosicoli and Brachyspira aalborgi, from a patient with ulcerative colitis.

We succeeded in the simultaneous isolation of Brachyspira (B.) aalborgi and B. pilosicoli from a patient with ulcerative colitis. B. pilosicoli grew quickly and formed colonies within 7 days, while the growth of B. aalborgi was very slow and took over 21 days. Simultaneous isolation of B. pilosicoli and B. aalborgi from a common specimen is generally recognized to be difficult, mainly due to differences in their growth requirements and the growth rates. However, we succeeded in isolating both species from a patient with ulcerative colitis and this is first evidence. The present results suggest that ulcerative colitis may be caused by simultaneous infection with B. pilosicoli and B. aalborgi.

Differentiation of Brachyspira spp. isolated from laying hens using PCR-based methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Avian intestinal spirochetosis (AIS), an important but neglected disease in laying hens, is caused by Brachyspira pilosicoli, B. intermedia, and B. alvinipulli. Poultry are also frequently colonized by putatively nonpathogenic species such as B. murdochii and B. innocens. We evaluated the differentiation of Brachyspira species by 3 methods: sequencing of the reduced nicotinamide adenine dinucleotide (NADH) oxidase gene ( nox), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a new multiplex (m)PCR targeting genes such as the tryptophanase A gene ( tnaA) and the p-aminobenzoyl-glutamate hydrolase subunit B gene ( abgB). Sequencing of 414 bp of the nox PCR amplification products generated from 41 pure cultures of avian Brachyspira isolates allowed presumptive species identification in 33 isolates with at least 99% identity in basic local alignment search tool analysis, including B. pilosicoli, B. intermedia, B. murdochii, B. innocens, and " B. pulli". MALDI-TOF MS analysis was found to be a reliable tool for differentiation after extension of the manufacturer's database. In the mPCR, all isolates identified as B. pilosicoli and B. intermedia were positive for abgB and tnaA, respectively. The mPCR might be very useful in detecting Brachyspira species in mixed cultures including not only nonpathogenic species, such as B. innocens, but also one of the AIS pathogens. We found that MALDI-TOF MS analysis combined with the mPCR targeting tnaA and abgB was suitable for the identification of avian isolates of B. pilosicoli and B. intermedia, 2 important agents of AIS.

Antimicrobial Susceptibility Patterns of Brachyspira Species Isolated in Taiwan.

Some members of the Brachyspira genus cause diseases such as swine dysentery (SD) and porcine intestinal (or colonic) spirochetosis. Severe economic losses are caused by decreased feed intake and increased feed conversion ratio, as well as costs associated with treatment and death. A loss of clinical efficacy of some antimicrobial agents authorized for treating SD has been observed in many countries. The aim of this study was to analyze the antimicrobial susceptibility of Brachyspira isolated from Taiwan and to investigate the mechanism of decreased susceptibility to macrolides. A total of 55 Brachyspira isolates obtained from the grower-finisher period were evaluated in this study. These isolates included B. hyodysenteriae (n = 37), B. murdochii (n = 11), B. pilosicoli (n = 5), B. intermedia (n = 1), and B. innocens (n = 1). Antimicrobial susceptibility testing was performed to examine 12 selected antimicrobial agents. The results showed that the 50% and 90% minimum inhibitory concentration (MIC) values of the tested macrolides were all >256 µg/ml. The MIC50 of lincomycin, tiamulin, carbadox, olaquindox, ampicillin, amoxicillin, doxycycline, oxytetracycline, and gentamicin were 32, 1, ≤0.125, ≤0.125, 0.5, 0.25, 2, 2, and 2 µg/ml. The genetic basis of the decreased susceptibility to tylosin and lincomycin in Brachyspira spp. was investigated and the results showed a possible connection to the mutations at position A2058 and G2032 of the 23S rRNA gene. These findings demonstrated that, in Taiwan, there may be a decrease in susceptibility of Brachyspira spp. to antimicrobials commonly used for the treatment of SD.

Occurrence of dysentery-like diarrhoea associated with Brachyspira suanatina infection on a German fattening pig farm.

The anaerobic intestinal spirochaete Brachyspira (B.) suanatina was first described in 2007 but since then no further isolates have been reported from pigs. Accordingly, when the species was validly published in 2016, the overall occurrence and clinical relevance in pigs were unknown. In a fattening farm in southern Germany, mucohaemorrhagic diarrhoea was observed in 60 per cent (750 animals) of the finisher pigs. A diagnostic workup including Brachyspira culture, Salmonella culture, Lawsonia intracellularis-specific, B. hyodysenteriae-specific and B. pilosicoli-specific multiplex PCR and postmortem examination of severely affected pigs was performed. Tests for Salmonella species, Lawsonia intracellularis and B. hyodysenteriae were all negative. Gross and microscopic lesions were in agreement with dysentery and spirochaetes could be demonstrated by silver staining in tissue samples of the caecum at the ileal papilla. B. suanatina was cultured from faeces or colon of all (five) animals sampled and identified using nox-RFLP, partial nox-gene-sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). According to the initial report from Scandinavia, B. suanatina can be isolated from birds and cross-species infection could be demonstrated infecting pigs with an avian isolate. Thus outdoor production as in the case presented here and international trade may pose a risk for infection of naive herds.

Characterization of Brachyspira communities from clinical cases of swine mucohaemorrhagic diarrhea through deep sequencing of the NADH oxidase (nox) gene.

Swine dysentery is traditionally associated with Brachyspira hyodysenteriae, but the re-emergence of Brachyspira-associated disease in North America associated with a novel causative species, B. hampsonii, is now a concern for swine producers. The pathogenesis of Brachyspira-associated disease is not completely understood, and it is not known whether mixed infections of Brachyspira spp. are important in disease development. Deep sequencing of partial sequences of the nox gene amplified with genus-specific primers was used to detect Brachyspira spp. in 55 fecal samples from clinical cases of mucohaemorrhagic diarrhea in pigs from Western Canada that had been identified as positive for one or more Brachyspira species using established diagnostic tests. Synthetic mixtures of Brachyspira genomic DNA were included in the study to define detection limits for the technique and identify biases in detection of different species. Multiple species were detected in all clinical cases for which sufficient nox sequence data were generated (n = 47), indicating that mixed species Brachyspira infections are common, although in most cases, one species accounted for at least half of the sequences identified. In all cases, the species detected in the original diagnostic investigation of each case was also detected by nox sequencing. Results from synthetic communities indicated that the method was highly reproducible, but also indicated potential PCR bias against B. hampsonii genomovar I. Deep sequencing of the nox gene target is a suitable method for simultaneous detection of multiple Brachyspira species in clinical case material that may offer advantages over current, more targeted diagnostic approaches for investigating the significance of mixed infections in disease development.

Isolation and antimicrobial susceptibility of Brachy -spira species from feces of layer chickens in Germany.

OBJECTIVE: Anaerobic spirochetes of the genus Brachyspira are important pathogens causing swine dysentery (Brachyspira [B.] hyodysenteriae) and porcine intestinal spirochetosis (B. pilosicoli, PIS). In addition, avian intestinal spirochetosis (AIS) is caused by B. pilosicoli, B. intermedia and B. alvinipulli. Despite the economic impact of AIS, the disease has not received appropriate attention in Germany. This study was aimed at identifying Brachyspira spp. in Germany and determining their antimicrobial susceptibility. MATERIAL AND METHODS: From 2009 to 2013, a total of 71 fecal swabs were obtained from clinically healthy layer hens from eight different commercial flocks. Brachyspira spp. culture was performed in trypticase soybean agar added with 5% sheep blood. Species determination was conducted by PCRs targeting the NADH-gen and the 16S rDNA or by nox-gene sequencing. Antimicrobial susceptibility to macrolides, lincosamides and pleuromutilins was tested by a microdilution assay. RESULTS: Brachyspira spp. were isolated from 40 (56.3%) swabs distributed over all eight flocks. In 26 cases, the following species were determined by PCR: B. pilosicoli (n = 16), B. intermedia (2), B. innocens (3), B. murdochii (1), mixtures of B. pilosicoli/B. intermedia (2), B. innocens/B. intermedia (1), B. innocens/B. murdochii (1). Remaining isolates were characterized by noxgene sequencing as B. "pulli" (n = 9), B. alvinipulli (3), B. intermedia (1) and as not identifiable (1). Antimicrobial susceptibility testing of 37 isolates revealed minimal inhibitory concentrations 90 (MIC90) of > 128 mg/l (tylosin), 64 mg/l (lincomycin), 8 mg/l (tiamulin) and 4 mg/l (valnemulin), respectively. Comparing to breakpoints applied to pigs, these values lie within the range of resistance. CONCLUSION: The demonstration of different Brachyspira spp., particularly B. pilosicoli, intermedia and alvinipulli in commercial layers, indicates the need of further research to assess their potential role in causing AIS in German poultry flocks. The increased antimicrobial resistance of Brachyspira spp. isolates to tylosin and pleuromutilins is likely associated with extensive use of these drugs in poultry medicine.

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.

Distribution and phylogeny of Brachyspira spp. in human intestinal spirochetosis revealed by FISH and 16S rRNA-gene analysis.

During six years as German National Consultant Laboratory for Spirochetes we investigated 149 intestinal biopsies from 91 patients, which were histopathologically diagnosed with human intestinal spirochetosis (HIS), using fluorescence in situ hybridization (FISH) combined with 16S rRNA gene PCR and sequencing. Aim of this study was to complement histopathological findings with FISH and PCR for definite diagnosis and species identification of the causative pathogens. HIS is characterized by colonization of the colonic mucosa of the human distal intestinal tract by Brachyspira spp. Microbiological diagnosis of HIS is not performed, because of the fastidious nature and slow growth of Brachyspira spp. in culture. In clinical practice, diagnosis of HIS relies solely on histopathology without differentiation of the spirochetes. We used a previously described FISH probe to detect and identify Brachyspira spp. in histological gut biopsies. FISH allowed rapid visualization and identification of Brachyspira spp. in 77 patients. In most cases, the bright FISH signal already allowed rapid localization of Brachyspira spp. at 400× magnification. By sequencing, 53 cases could be assigned to the B. aalborgi lineage including "B. ibaraki" and "B. hominis", and 23 cases to B. pilosicoli. One case showed mixed colonization. The cases reported here reaffirm all major HIS Brachyspira spp. clusters already described. However, the phylogenetic diversity seems to be even greater than previously reported. In 14 cases, we could not confirm HIS by either FISH or PCR, but found colonization of the epithelium by rods and cocci, indicating misdiagnosis by histopathology. FISH in combination with molecular identification by 16S rRNA gene sequencing has proved to be a valuable addition to histopathology. It provides definite diagnosis of HIS and allows insights into phylogeny and distribution of Brachyspira spp. HIS should be considered as a differential diagnosis in diarrhea of unknown origin, particularly in patients from risk groups (e.g. patients with colonic adenomas, inflammatory polyps, inflammatory bowel disease or HIV infection and in men who have sex with men).

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 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.