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Journal ArticleDOI

Neither Hippurate-negative Brachyspira pilosicoli nor Brachyspira pilosicoli Type Strain Caused Diarrhoea in Early-weaned Pigs by Experimental Infection

31 Dec 2005-Acta Veterinaria Scandinavica (BioMed Central)-Vol. 46, Iss: 4, pp 257-267

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.

AbstractA 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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Citations
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Journal ArticleDOI
TL;DR: This investigation shows that infections with weakly haemolytic Brachyspira spp.
Abstract: The prevalence of infections with different Brachyspira species was assessed in 202 pigs with various chronic herd problems using different methods. Twenty-seven pigs (13.4%) were positive for Brachyspira spp. with at least one of the methods used. The highest number of positives was identified with mucosal scraping-PCR (23), followed by PET-PCR (22) and bacteriological–biochemical analysis (15). With the exception of three cases of B. pilosicoli infections, only weakly pathogenic Brachyspira species were identified. The majority was B. murdochii, followed by B. innocens and B. intermedia. Concurrent infections with two or more Brachyspira species were common and accounted for 37.1% of the total. Presence of weakly haemolytic Brachyspira was associated with wasting and diarrhoea in a number of cases. This investigation shows that infections with weakly haemolytic Brachyspira spp. may contribute to colonic pathology in pigs with chronic herd problems and that mixed infections seem to occur more frequently than previously noticed.

37 citations


Cites background from "Neither Hippurate-negative Brachysp..."

  • ..., 1998) and on the other hand biochemical typing is not designed to identify multiple infections (Fellström et al., 1996; Fossi et al., 2005; Råsbäck et al., 2005)....

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  • ...…the detection of more than one Brachyspira sp. in culture can be complicated by the overgrowth of one of the species (Møller et al., 1998) and on the other hand biochemical typing is not designed to identify multiple infections (Fellström et al., 1996; Fossi et al., 2005; Råsbäck et al., 2005)....

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Journal ArticleDOI
TL;DR: The spirochete, here named “Candidatus Treponema suis,” was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.
Abstract: Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantiiT (90.1%). The spirochete, here named “Candidatus Treponema suis,” was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa.

18 citations


Cites background or methods from "Neither Hippurate-negative Brachysp..."

  • ...The tissue sections were first hybridized with an oligonucleotide probe S-GLeptospira-1414-a-A-18, originally designed to target the Leptospira interrogans group (4), and subsequently with S-STreponema-0833-a-A-18, targeting the novel “Candidatus Treponema suis” (this study)....

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  • ...However, upon both histopathological and in situ hybridization examination of all 23 pigs, an unknown species of spirochetes rather than the challenged bacteria was found to have invaded the colonic mucosa of several of the pigs, including the control pigs (4)....

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  • ...pilosicoli strains (Br1622 and P43/6/8, respectively) and eight uninfected control pigs (4)....

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  • ...Treponema suis” were found to be longer (6 to 11 m) than those of other Treponema species (4 to 8 m) previously found in pigs (3, 4, 8)....

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  • ...Treponema suis” revealed 10 to 14 flagella (4)....

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Journal ArticleDOI
TL;DR: The current paper describes the methodological aspects of commercially available laser microdissection instruments and representative examples that demonstrate the advantages of this method for resolving a variety of issues in microbiology.
Abstract: Laser microdissection is a method that allows for the isolation of homogenous cell populations from their native niches in tissues for downstream molecular assays. This method is widely used for genomic analysis, gene expression profiling and proteomic and metabolite assays in various fields of biology, but it remains an uncommon approach in microbiological research. In spite of the limited number of publications, laser microdissection was shown to be an extremely useful method for studying host-microorganism interactions in animals and plants, investigating bacteria within biofilms, identifying uncultivated bacteria and performing single prokaryotic cell analysis. The current paper describes the methodological aspects of commercially available laser microdissection instruments and representative examples that demonstrate the advantages of this method for resolving a variety of issues in microbiology.

11 citations


Dissertation
01 Jan 2009
TL;DR: The work described in this thesis was concerned with identifying the prevalence and risk factors associated with colonisation by the intestinal spirochaete Brachyspira pilosicoli in humans and Indonesians either living temporarily in Perth or as long term residents in urban and rural areas of Bali, Indonesia.
Abstract: The work described in this thesis was concerned with identifying the prevalence and risk factors associated with colonisation by the intestinal spirochaete Brachyspira pilosicoli in: humans: long term residents of Perth, Western Australia (WA) and Indonesians either living temporarily in Perth or as long term residents in urban and rural areas of Bali, Indonesia, animals: domestic animals including alpacas, birds, cattle, cats, chickens, dogs, doves, ducks, goats, horses, pigs, and sheep (housed at a wide variety of places around Perth), and a range of wild animals housed in various Zoos and wildlife centres in WA This study shows that for humans: • Brachyspira pilosicoli was significantly more prevalent in Indonesians of all sub groups, be they temporary residents of Perth (94% - 216 faecal samples from 180 individuals), or long term residents of Indonesia (126% - 992 faecal samples from 617 individuals) compared with long term residents of Australia living mainly in Perth (02% of 766 sampled), even in those with gastrointestinal complaints This suggests a relationship between a high prevalence of B pilosicoli and living in Indonesia; • In Bali, B pilosicoli was significantly more prevalent in the impoverished urban area of Sesetan (203-234%) where the husbandry of pigs is poor and effluent treatment is non-existent compared to four traditional farming villages (Badung, Karang Suwung, Melinggih, Payangan Desa) (33-226%) In the latter villages effluent and drainage is better and there is less likely to be contamination of drinking water • There was no significant association between the presence of B pilosicoli and the presence of clinical symptoms including headaches, abdominal pains, diarrhoea, joint/muscular pain and constipation • Amongst Indonesians living in Indonesia, there was no significant difference in the prevalence of B pilosicoli between people with and without contact with animals and between farmers and other occupational groups • Indonesians visiting Perth who were positive for B pilosicoli originated from nine cities and five main islands in Indonesia This suggests that B pilosicoli is endemic throughout Indonesia • Strain typing of isolates of B pilosicoli showed that they were genetically heterogenous and did not show any consistent pattern with respect to geographical location, family of origin or disease status Isolates from the same individual were sometimes unrelated, suggesting the probability of re-infection with another strain between the samplings • Some households (~7%) had more than one member positive for B pilosicoli Strain analysis suggested transmission between family members, and this could be due to either faecal-oral transmission, or from a common external source, such as contaminated water • B pilosicoli was cultured from only 02% of Australians This low prevalence may be a result of little or no exposure to B pilosicoli due to good personal hygiene and environmental sanitation • B pilosicoli strain H1b and H171 that were isolated from healthy Indonesians were able to colonise mice and day-old chickens, and induced clinical signs of pasty faeces in the latter Histological sections showed mild typhlitis and typical end-on attachment of B pilosicoli to the caecal epithelial mucosa of the chickens This finding suggests that the human isolates had pathogenic potential This study showed that for animals investigated: • Intestinal spirochaetes were cultured from 464% (13/28) of bilbies with 143% (4/28) positive for B pilosicoli Spirochaetes were also cultured from the faeces of two Western Barred bandicoots and one (12%) kangaroo • Intestinal spirochaetes were not isolated from any alpacas, cattle, goats, horses, pigs, and sheep but were detected in 405% of ducks, 143% of chickens, 149% of ostriches and 15% of cats • Few pets that are commonly kept in households (dogs, cats and aviary birds) were colonised, suggesting that they are not an important focus of B pilosicoli infection in Australia

3 citations


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: 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.
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"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.
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Journal ArticleDOI
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.

592 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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Journal ArticleDOI
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.

233 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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