Neither Hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli Type Strain Caused Diarrhoea in Early-weaned Pigs by Experimental Infection
TL;DR: The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.
Abstract: A hippurate-negative biovariant of Brachyspira pilosicoli (B. pilosicolihipp-) is occasionally isolated in diarrhoeic pigs in Finland, often concomitantly with hippurate-positive B. pilosicoli or Lawsonia intracellularis. We studied pathogenicity of B. pilosicolihipp- with special attention paid to avoiding co-infection with other enteric pathogens. Pigs were weaned and moved to barrier facilities at the age of 11 days. At 46 days, 8 pigs were inoculated with B. pilosicolihipp- strain Br1622, 8 pigs were inoculated with B. pilosicoli type strain P43/6/78 and 7 pigs were sham-inoculated. No signs of spirochaetal diarrhoea were detected; only one pig, inoculated with P43/6/78, had soft faeces from day 9 to 10 post inoculation. The pigs were necropsied between days 7 and 23 after inoculation. Live pigs were culture-negative for Brachyspira spp., but B. pilosicolihipp- was reisolated from necropsy samples of two pigs. The lesions on large colons were minor and did not significantly differ between the three trial groups. In silver-stained sections, invasive spirochaetes were detected in colonic mucosae of several pigs in all groups. Fluorescent in situ hybridisation for genus Brachyspira, B. pilosicoli and strain Br1622 was negative. However, in situ detection for members of the genus Leptospira was positive for spirochaete-like bacteria in the colonic epithelium of several pigs in both infected groups as well as in the control group. L. intracellularis, Salmonella spp., Yersinia spp. and intestinal parasites were not detected. The failure of B. pilosicoli strains to cause diarrhoea is discussed with respect to infectivity of the challenge strains, absence of certain intestinal pathogens and feed and management factors.
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Dissertation•
01 Jan 2010
TL;DR: In vitro studies have shed new light onto the pathogenic processes that are involved in intestinal spirochaetosis caused by B. pilosicoli infections and revealed that as well as chemotaxis to mucin components, “viscotaxis” is involved in the attraction to mucIn.
Abstract: Brachyspira pilosicoli is an intestinal spirochaete that colonizes the large intestine of a variety of species of birds and animals, including human beings. Colonization can lead to local inflammation and to diarrhoea in a condition known as “intestinal spirochaetosis”. This infection has been described in many countries throughout the world. In the colonization process the bacterium must cross the thick mucus blanket overlaying the colonic epithelium. Characteristically, B. pilosicoli then attaches by one cell end to the underlying epithelium, forming a dense “false brush border”. The mechanisms involved in moving through the mucus layer, attaching to enterocytes and inducing local cellular damage are poorly understood. The lack of in vitro models to study these events has been a major constraint to understanding the pathogenesis of B. pilosicoli infections.
The work described in this thesis deals with i) the development of an in vitro model of spirochaete attachment by using cells in suspension (erythrocytes) and cell monolayers (Caco-2), ii) the attraction of B. pilosicoli to mucin, and iii) the effects of norepinephrine exposure on expression of virulence traits by B. pilosicoli.
Attachment assays conducted with erythrocytes from different species at different ratios and time intervals identified one human isolate (WesB) that adhered to goose and chicken erythrocyte at a 1:1000 ratio. This same strain, and an isolate from a pig (95/1000) also attached to Caco-2 cells. Transmission and scanning electron microscopy confirmed that the attachment resembled the in vivo situation. Exposure of the Caco-2 cells to B. pilosicoli resulted in actin rearrangements, damaged cell junctions and apoptosis. Caco-2 cells that were colonized with B. pilosicoli also demonstrated a significant up-regulation of interleukin-1s (IL-1s) and IL-8 expression, helping to confirm that the spirochaetes were inducing pathological changes in the cultured cells. Treatment of the monolayers with B. pilosicoli sonicates caused significant up-regulation of IL-1s, TNF-α, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not altered cytokine expression. Hence Il-8 expression was specifically associated with exposure to live B. pilosicoli cells.
For mucin attraction, 15 B. pilosicoli strains isolated from humans, pigs, chickens and dogs, and a control strain of Brachyspira hyodysenteriae, were analysed for their ability to enter solutions of hog gastric mucin in an in vitro capillary tube assay. Attraction started in a 2 % mucin solution, and then increased with increasing concentrations to peak at around 6 - 8 % mucin. Attraction varied from strain to strain. B. pilosicoli strain 95/1000 and B. hyodysenteriae strain B204 also were attracted to viscous solutions of polyvinylpyrillodone (PVP), in a manner mirroring the response to mucin. This suggested that as well as chemotaxis to mucin components, “viscotaxis” is involved in the attraction to mucin.
Finally, exposure of B. pilosicoli to norepinephrine enhanced the attachment to Caco-2 cells, chemotactic response to mucin, and spirochaete growth. Taken together, these in vitro studies have shed new light onto the pathogenic processes that are involved in intestinal spirochaetosis caused by B. pilosicoli.
3 citations
Cites background from "Neither Hippurate-negative Brachysp..."
...In an experimental pig infection trial, B. pilosicoli (strain P43/6/78) crossed the epithelial cell layer into the lamina propria of the caecum and colon (Fossi et al 2005)....
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TL;DR: This is the first report of whole genome analysis of clinical isolates from individuals with colonic spirochaetosis, and it is shown that 16S amplicon sequencing using standard primers for human microbiota studies fail to detect Brachyspira due to primer incompatibility.
Abstract: Colonic spirochaetosis, diagnosed based on the striking appearance in histological sections, still has an obscure clinical relevance and only 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. In these individuals, 17 (2.3 %) had colonic spirochaetosis, which was associated with eosinophilic infiltration and a three-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 spirochaetosis we successfully isolated, cultured and performed whole genome sequencing of in total 17 isolates including the Brachyspira aalborgi type strain 513AT. Also, 16S analysis of the mucosa-associated microbiota was performed in the cases and non-spirochaetosis controls. This is the first report of whole genome analysis of clinical isolates from individuals with colonic spirochaetosis. We found one isolate to be of the species Brachyspira pilosicoli and all remaining isolates were of the species Brachyspira aalborgi. Besides displaying extensive genetic heterogeneity, the isolates harboured 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 fail to detect Brachyspira due to primer incompatibility. This failure to detect colonic spirochaetosis should be taken into consideration in project design and interpretation of gastrointestinal tract microbiota in population-based and clinical settings.
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References
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TL;DR: The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface.
Abstract: The ARB (from Latin arbor, tree) project was initiated almost 10 years ago. The ARB program package comprises a variety of directly interacting software tools for sequence database maintenance and analysis which are controlled by a common graphical user interface. Although it was initially designed for ribosomal RNA data, it can be used for any nucleic and amino acid sequence data as well. A central database contains processed (aligned) primary structure data. Any additional descriptive data can be stored in database fields assigned to the individual sequences or linked via local or worldwide networks. A phylogenetic tree visualized in the main window can be used for data access and visualization. The package comprises additional tools for data import and export, sequence alignment, primary and secondary structure editing, profile and filter calculation, phylogenetic analyses, specific hybridization probe design and evaluation and other components for data analysis. Currently, the package is used by numerous working groups worldwide.
6,757 citations
"Neither Hippurate-negative Brachysp..." refers methods in this paper
...The strain-specific probe for Br1622, the specific probe for Treponema (pallidum group) and the genus-specific probes for Leptospira and Borrelia were selected using ARB software (Strunk et al. 2000)....
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TL;DR: Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry and the intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.
Abstract: Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA. Images
4,110 citations
Book•
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01 Jul 1952
TL;DR: In this article, the characteristics of a variety of diseases of swine and methods for their prevention and treatment are described, as well as methods to detect and treat these diseases in swine.
Abstract: Describes the characteristics of a variety of diseases of swine, and methods for their prevention and treatment.
1,722 citations
TL;DR: The Oligonucleotide Probe Database (OPD) is designed and modified to include multiple probe versions and also to provide additional identifying information, and a method of standardizing the nomenclature for oligon nucleotide probes and PCR primers that is both unambiguous and informative is suggested.
Abstract: The use of oligonucleotide hybridization probes and PCR primers has become widespread in microbial ecology and environmental microbiology (for reviews, see references 3, 5, 7, 17, and 21), and descriptions of probe applications are abundant in the literature. We have encountered, however, a number of difficulties when relying on the literature for information on probes and primers: (i) probe design, characterization, and application data are scattered throughout the literature and therefore are not easily available; (ii) probe nomenclature is not standardized, making it difficult to recognize a particular probe and evaluate results obtained with that probe; (iii) probes are often designed empirically and used without thorough experimental characterization, making it difficult to interpret experimental results; and (iv) information on the application of individual probes is often not published in detail in the original probe description since the value of some data becomes apparent only as a result of observations made subsequent to publication (e.g., hybridization buffer composition, formamide concentration, membrane supplier and lot number, target group specificity). We designed the Oligonucleotide Probe Database (OPD) to address these concerns. The OPD centralizes information related to the design and use of oligonucleotide probes and PCR primers. The database was originally designed in Microsoft Access Version 2.0 and then converted to Hypertext Transfer Markup Language with PERL scripts. The current data set contains 96 hybridization probes and PCR primers used in microbial ecology and environmental microbiology, published by the authors as well as from direct on-line submissions to OPD. The majority of the probes in the current data set target rRNA, but the database is designed to accommodate probes targeting other gene families. For each probe or primer, the information in the OPD includes design and characterization data important for probe and primer use, including a standardized name, probe sequence, nucleotide position within the target gene, optimal hybridization and wash conditions (or annealing conditions for PCR primers), intended target group, experimentally validated target group specificity, and original citations. Much of the experimental data available in the database were not included in the original publications describing the probes. Standardization of oligonucleotide probe nomenclature. A source of much confusion and frustration during the use of probes or PCR primers designed and characterized in different laboratories has been the absence of a standardized probe nomenclature. Stahl and Amann (18) have previously attempted to address this problem for phylogenetic probes with a nomenclature consisting of three to five letters representing phylogenetic specificity, followed by a number indicating the 59 position on the rRNA complementary to the 39 end of an antisense probe or PCR primer or identical to the 59 end of a sense primer. Limitations to this nomenclature system are apparent when several versions of a probe that have the same target specificity and nucleotide position exist. We modified the nomenclature originally utilized by Stahl and Amann to include multiple probe versions and also to provide additional identifying information. We suggest a method of standardizing the nomenclature for oligonucleotide probes and PCR primers that is both unambiguous and informative. The name for an oligonucleotide probe consists of seven components combined sequentially. These components are discussed below. An example demonstrating construction of probe nomenclature for a small-subunit rRNA-targeted probe is given in parentheses.
608 citations
"Neither Hippurate-negative Brachysp..." refers background in this paper
...1 According to the Oligonucleotide Probe Database (OPD) nomenclature (Alm et al. 1996)....
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TL;DR: DNA-DNA relative reassociation experiments in which the S1 nuclease method was used revealed that intestinal spirochete strain P43/6/78T was related to, but was genetically distinct from, both S. hyodysenteriae B78T and S. innocens B256T, and it is proposed that strain P 43/ 6/78 should be designated as the type strain of a new species, Serpulina pilosicoli.
Abstract: Phenotypic and genetic traits of porcine intestinal spirochete strain P43/6/78T (= ATCC 51139T) (T = type strain), which is pathogenic and weakly beta-hemolytic, were determined in order to confirm the taxonomic position of this organism and its relationships to previously described species of intestinal spirochetes. In BHIS broth, P43/6/78T cells had a doubling time of 1 to 2 h and grew to a maximum cell density of 2 x 109 cells per ml at 37 to 42°C. They hydrolyzed hippurate, utilized D-glucose, D-fructose, sucrose, D-trehalose, D-galactose, D-mannose, maltose, N-acetyl-D-glucosamine, D-glucosamine, pyruvate, L-fucose, D-cellobiose, and D-ribose as growth substrates, and produced acetate, butyrate, ethanol, H2, and CO2 as metabolic products. They consumed substrate amounts of oxygen and had a G+C content (24.6 mol%) similar to that of Serpulina hyodysenteriae B78T (25.9 mol%). Phenotypic traits that could be used to distinguish strain P43/6/78T from S. hyodysenteriae and Serpulina innocens included its ultrastructural appearance (each strain P43/6/78T cell had 8 or 10 periplasmic flagella, with 4 or 5 flagella inserted at each end, and the cells were thinner and shorter and had more pointed ends than S. hyodysenteriae and S. innocens cells), its faster growth rate in liquid media, its hydrolysis of hippurate, its lack of β-glucosidase activity, and its metabolism of D-ribose. DNA-DNA relative reassociation experiments in which the S1 nuclease method was used revealed that P43/6/78T was related to, but was genetically distinct from, both S. hyodysenteriae B78T (level of sequence homology, 25 to 32%) and S. innocens B256T (level of sequence homology, 24 to 25%). These and previous results indicate that intestinal spirochete strain P43/6/78T represents a distinct Serpulina species. Therefore, we propose that strain P43/6/78 should be designated as the type strain of a new species, Serpulina pilosicoli.
236 citations
"Neither Hippurate-negative Brachysp..." refers background in this paper
...…spirochaetosis (PIS) in weaned pigs is caused by an anaerobic, weakly ß-haemolytic spirochaete, Brachyspira pilosicoli (Taylor et al. 1980, Trott et al. 1996), and it occurs worldwide (Duhamel 1998, Møller et al. 1998, Barcellos et al. 2000, Heinonen et al. 2000, de Arriba et al. 2002,…...
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...This strain isolated and reported by Taylor et al. in 1980, was later deposited in the American Type Culture Collection as a type strain of species B. pilosicoli (Trott et al. 1996)....
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