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Bacterial vaginosis (BV) is characterized by a reduction in Lactobacillus (L.) spp. abundance and increased abundance of facultative anaerobes, such as Gardnerella spp. BV aetiology is not fully understood; however, bacteriophages could play a pivotal role in the perturbation of the vaginal bacterial community. We investigated the vaginal viral community, including bacteriophages and the association to the bacterial community and BV-status. Vaginal samples from 48 patients undergoing IVF treatment for non-female factor infertility were subjected to metagenomic sequencing of purified virus-like particles. The vaginal viral community was characterized and correlated with the BV-status by Nugent score, bacterial community, structure, and the presence of key vaginal bacterial species. The majority of identified vaginal viruses belonged to the class of double-stranded DNA bacteriophages, with eukaryotic viruses constituting 4% of the total reads. Clear links between the viral community composition and BV ( q = 0.006, R = 0.26) as well as the presence of L. crispatus ( q = 0.001, R = 0.43), L. iners , Gardnerella spp., and Atopobium vaginae were found ( q < 0.002, R > 0.15). The eukaryotic viral community also correlated with BV-status ( q = 0.018, R = 0.20). In conclusion, the vaginal virome was clearly linked with bacterial community structure and BV-status.

Potato fiber is a side product in starch manufacturing rich in dietary fibers such as pectin, cellulose, hemicellulose and resistant starch. So far, the beneficial properties of potato fiber have been poorly characterized. This study investigated the effect of FiberBind 400, a commercial potato fiber product, on survival of probiotic Lactobacillus strains at simulated gastric conditions and on the composition and metabolic activity of the gut microbiota, using the TIM-2 colon model. Resistant starch and native starch from potato were used as reference substrates. FiberBind 400 had an ability to improve survival of the four tested strains, Lactobacillus fermentum PCC®, L. rhamnosus LGG®, L. reuteri RC-14® and L. paracasei F-19® in a strain-dependent way. The highest effect was observed for L. fermentum PCC® and L. rhamnosus LGG®. The effect of starches on bacterial survival was insignificant. Composition of the fecal microbiota in TIM-2 fermentations was assessed by high-throughput sequencing of 16S rRNA gene amplicon. Fermentation of FiberBind 400 resulted in more diverse microbial communities compared to starches. Changes in microbial abundances specifically mediated by FiberBind 400, included increases in the genera Lachnospira, Butyrivibrio, Mogibacterium, Parabacteroides, Prevotella and Desulfovibrio, and the species B. ovatus, as well as decreases in Ruminococcus torques and unassigned Ruminococcus spp. Shifts in other bacterial populations, such as increased abundances of Oscillospira, Enterococcus, Bacteroidales, Citrobacter, along with reduction of Roseburia, Ruminococcus, and Faecalibacterium prausnitzii were not significantly different between the substrates. Cumulative production of individual short-chain fatty acids was similar between potato fiber and starches. The study demonstrated that FiberBind 400 had a potential to protect probiotic Lactobacillus strains during the passage through the gastrointestinal tract and selectively modulate the gut bacterial populations. This knowledge can support application of potato fiber as a functional food ingredient with added biological benefits., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The microbiota has been shown to have the potential to affect appetite and blood lipids positively in animal studies. We investigated if four weeks supplementation with Lactobacillus paracasei subsp. paracasei L. casei W8® (L. casei W8) had an effect on subjective appetite sensation, ad libitum energy intake, glucagon-like peptide 1 (GLP-1), glucose and insulin response in humans. Secondarily, we explored potential effects on blood lipids, fatty acids and stearoyl-CoA desaturase-1 (SCD1) activity in humans as well as SCD1 expression in piglets given L. casei W8 for two weeks. 64 healthy participants completed the double-blinded, randomised, controlled, parallel four weeks study with supplementation of L. casei W8 (1010 cfu) or placebo capsules. A meal test was conducted before and after the intervention, where subjective appetite, ad libitum energy intake, GLP-1, glucose and insulin response were measured. Additionally fasting blood lipids and fatty acids concentrations were measured. Sixteen piglets were randomised into two groups: L. casei W8 (1010 cfu/day) as top dressing on morning fed or no treatment. After two weeks piglets were sacrificed and tissue from ileum, jejunum and skeletal muscle were sampled for mRNA analyses of SCD1 expression. Compared to placebo, L. casei W8 did not affect appetite, ad libitum energy intake, GLP-1, glucose and insulin response and total, high-density or low-density lipoprotein cholesterol levels after four weeks intervention. Triacylglycerol decreased in the L. casei W8 group compared to placebo at week 4 (P=0.03). The C16:1n-7/C16:0 ratio, reflecting SCD1 activity, tended to decrease when having L. casei W8 (P=0.06) compared to placebo. Muscle SCD1 expression decreased in piglets supplemented with L. casei W8 compared to control. In conclusion, supplementation with L. casei W8 did not affect appetite parameters, glucose or insulin responses; but appear to be able to lower triacylglycerol levels, possibly by reducing its production.

Background: Probiotic bacteria have been shown to have various effects on the microbiota; this may also affect appetite and may help promote weight loss and maintenance., Objective: This study was conducted to investigate the effect of Lactobacillus paracasei subsp paracasei L. casei W8 (L. casei W8) on glucagon-like peptide-1 (GLP-1) responses in an isolated pig intestine, in piglets and postprandially in humans. Additionally, the effect on subjective appetite, ad libitum energy intake, and glucose and insulin responses in humans was investigated., Design: Piglets were fed with probiotics for 2 weeks and the effect on glucagon encoding gene (GCG) was investigated. An isolated pig intestine was perfused with L. casei W8 and the GLP-1 response was measured. Twenty-one subjects completed a randomized, controlled, crossover study with three arms. Each participant completed 3 test days testing the effect of low dose (LD) (10(9) CFU), high dose (HD) (10(10) CFU) L. casei W8 or placebo capsule. Subjective appetite was assessed before an ad libitum lunch was served. GLP-1, insulin and glucose concentrations were analyzed., Results: Two weeks of treatment of piglets with L. casei W8 resulted in an increase in GCG expression compared to control animals (P<.05). L. casei W8 increased the GLP-1 response in the isolated pig intestine. In humans, L. casei W8 had an overall effect on energy intake (P=0.03), but no effects on subjective appetite sensation, overall glucose and insulin response and on GLP-1 release were observed (P>0.1)., Conclusion: The probiotic bacteria L. casei W8 appears to lower food intake acutely, but the underlying mechanisms are not understood., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Consumption of prebiotics may modulate gut microbiota, subsequently affecting the bacterial composition, metabolite profile, and human health. Previous studies indicate that also changes in intestinal integrity may occur. In order to explore this further we have investigated the effect of the putative prebiotic xylo-oligosaccharides (XOS) on the gut microbiota and intestinal integrity in male Wistar rats. As changes in intestinal integrity may be related to the expected bifidogenic effect of XOS, we additionally addressed effects of supplementation with a commensal Bifidobacterium pseudolongum (BIF) isolated from the same breed of laboratory rats., Results: Changes in faecal and caecal bacterial composition determined by 16S rRNA gene sequencing and quantitative PCR for selected bacterial groups revealed that the overall bacterial composition did not differ markedly between the control (CON), XOS, and BIF groups, when correcting for multiple comparisons. However as hypothesised, the relative abundance of Bifidobacterium spp. was increased in XOS-fed rats as compared to CON in faecal samples after the intervention. Also Lactobacillus spp. was increased in both the XOS and BIF groups in caecum content compared to CON. Intestinal permeability determined in vivo by FITC-dextran permeability and in vitro using extracted caecum water in trans-epithelial resistance (TER) assay showed no effect on intestinal integrity in either the XOS or the BIF groups. However, the expression of occludin, which is part of the tight junction complex, was increased in the XOS group compared to the CON group., Conclusions: Supplementation with XOS or a commensal Bifidobacterium pseudolongum had very limited effects on intestinal integrity in rats as only significant change in expression of a single tight junction protein gene was found for the XOS group.

Background & Aims: The aim of this study was to investigate the spectrum of colonic microflora in patients with colitis and if this could be altered with one month's treatment with synbiotics., Methods: This was a pilot study in which patients were randomised to either receive a synbiotics preparation for a month and then "crossed over" to receive a placebo, or alternatively to receive the placebo first followed in the second month by synbiotic. Stool samples were collected on entry into the study and then at the end of first and second months respectively. Colonic microflora was measured by terminal restriction fragment length polymorphism technique. Quantitative PCR was used to determine the concentration of individual species., Results: Sixteen patients completed the study of whom 8 had Crohn's colitis and 8 had ulcerative colitis. Their median age was 62 (IQR 50-65) years. An average of 22 terminal restriction fragments (T-RF's) was identified in each patient. Dice cluster analysis showed that each patient had a unique microbial composition which did not change significantly at different time points in the study, irrespective of whether they had probiotics or the placebo. Probiotic organisms were identified in stool samples but did not alter overall spectrum of microflora. In this pilot study we were unable to identify any specific characteristics related to nature of colitis., Conclusions: This study suggests that there is no difference in colonic microflora between patients with Crohn's or Ulcerative colitis and that the spectrum of bacteria was not altered by synbiotic administration., (Copyright © 2013 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.)

Mammals live in a homeostatic symbiosis with their gastrointestinal microbiota. The mammalian host provides the microbiota with nutrients and a stable environment; whereas the microbiota helps shaping the host's gut mucosa and provides nutritional contributions. Microorganisms start colonizing the gut immediately after birth followed by a succession of populations until a stable, adult microbiota has been established. However, physiological conditions differ substantially among locations in the gut and determine bacterial density and diversity. While Firmicutes and Bacteroidetes dominate the gut microbiota in all mammals, the bacterial genera and species diversity is huge and reflects mammalian phylogeny. The main function of the gastrointestinal epithelium is to absorb nutrients and to retain water and electrolytes, yet at the same time it is an efficient barrier against harmful compounds and microorganisms, and is able to neutralize antagonists coincidentally breaching the barrier. These processes are influenced by the microbiota, which modify epithelial expression of genes involved in nutrient uptake and metabolism, mucosal barrier function, xenobiotic metabolism, enteric nervous system and motility, hormonal and maturational responses, angiogenesis, cytoskeleton and extracellular matrix, signal transduction, and general cellular functions. Whereas such effects are local at the gut epithelium they may eventually have systemic consequences, e.g. on body weight and composition.

Aims: To determine if orally ingested Bacillus spores used as probiotics or direct-fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract., Methods and Results: Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore-feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4-6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid-colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2.14-fold compared to time zero., Conclusions: The experiments showed that 70-90% of dietary-supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract., Significance and Impact of the Study: A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.

Pigs with and without naturally occurring Lawsonia intracellularis infection were fed diets with different texture. In a previous study from 79 pig herds using a similar feeding on pelleted or non-pelleted form showed that the non-pelleted diet was associated with a reduced prevalence of L. intracellularis. In this study a mechanistic approach was taken for explaining and testing this observation by studying the microbiota and the occurrence of L. intracellularis in the distal ileum of 54 pigs by terminal restriction fragment length polymorphism (T-RFLP) analysis, Real-Time PCR and in situ hybridization. The texture of the diet influenced the microbiota, and from a quantitative discriminative analysis of the terminal restriction fragments (T-RFs) of ileum samples it was deduced that Clostridium spp. and Lactobacillus spp. were associated with the non-pelleted diet and Streptococcus spp. with the pelleted diet. In experimentally infected pigs it was verified that 89bp and 90bp sized T-RFs (HhaI) from ileum represented L. intracellularis. The non-pelleted diet seemed to reduce the relative amount of L. intracellularis in the total microbiota of the ileum, but the number of pigs detected positive with L. intracellularis by Real-Time PCR was not influenced. The five pigs with highest L. intracellularis content showed T-RFs that were not present in profiles from less or non-infected pigs, which may indicate that some bacterial species were associated with L. intracellularis infection.

The immediate post-weaning period is often associated with gut malfunction and diarrhoea for young pigs. Administration of antimicrobials remains an effective way to control weaning diarrhoea but it remains unclear how they affect gut physiology and microbiology although this is a prerequisite for being able to devise better alternatives. Hence, for 7 d we treated pigs, weaned at 24 d of age, with a combination of amoxicillin (25 mg/kg feed and injection of 8.75 mg/kg body weight per 12 h) and ZnO (2.5 g/kg feed). The pigs treated with antimicrobials (n 11) showed no signs of gut malfunction at any time, whereas untreated weaned controls (n 11) developed clinical diarrhoea. The antimicrobial treatment resulted in a higher daily weight gain compared with weaned controls (101 v. -44 g/d, P < 0.0001), whereas both groups had a similar degree of villous atrophy compared with unweaned 24-d-old controls (n 8; P < 0.05). The antimicrobial treatment gave a dramatic reduction in small intestinal microbial diversity, and specifically prevented tissue colonization with Escherichia coli compared with weaned controls. Further, the antimicrobial treatment improved amylase, trypsin and small intestinal aminopeptidase A and N activities (all P < 0.05). Specifically for the colon, the antimicrobial treatment was associated with reduced tissue weight ( -23 %, P < 0.05), reduced concentration of SCFA (P < 0.05), and increased mucosal goblet cell area (P < 0.0001) compared with weaned controls. We conclude that the beneficial effects of antimicrobials are mediated not only through reduction in intestinal bacterial load, but also through a stimulation of protein digestive function and goblet cell density.

Background: The aim of this study was to investigate whether it is possible to modulate gut microflora and preserve intestinal barrier function during elective colorectal surgery by using combinations of oral antibiotics, synbiotics and mechanical bowel preparation (MBP)., Methods: Ninety-two patients were randomly assigned to one of four groups. Group 1 had MBP only, group 2 had neomycin + MBP, group 3 had synbiotics + neomycin + MBP, and group 4 had synbiotics + neomycin but no MBP. Changes in gut microflora were assessed by culturing nasogastric aspirates and polymerase chain reaction-denaturing gradient gel electrophoresis of faecal samples. Intestinal barrier function was determined by microbiological confirmation of bacterial translocation and measurement of intestinal permeability. The inflammatory response was monitored by measurement of serum C-reactive protein and interleukin 6, and septic morbidity was recorded prospectively., Results: Four patients were excluded owing to protocol violation, leaving 24 patients in group 1, 22 in group 2, 20 in group 3 and 22 in group 4 for analysis. There was a significant decrease in Enterobacteriaceae in group 3 compared with the other groups. Group 3 had a significantly lower incidence of translocation after bowel mobilization (P < 0.001). There was no significant difference between the groups in intestinal permeability, inflammatory response or septic morbidity., Conclusion: The combination of MBP, neomycin and synbiotics reduces the prevalence of faecal Enterobacteriaceae and bacterial translocation; however, this was not associated with a reduction in inflammatory response or septic morbidity in this study. Larger trials are needed before a change in practice can be recommended.

A multiplex real-time PCR assay was developed for absolute quantification in animal feed of Bacillus subtilis CH201 and Bacillus licheniformis CH200 spores, which constitute the viable component of the microbial growth promoter, BioPlus 2B. Spores were lysed using a bead-beating protocol. DNA was extracted and purified from the lysates with the Qiagen DNeasy Plant Kit. Two standard curves for absolute quantification were made and tested. Standard curve-1 was made from feed samples spiked with BioPlus 2B, while standard curve-2 was made from serially diluted DNA extracted from BioPlus 2B powder. Feed samples supplemented with BioPlus 2B were quantified using both standard curves. The detection limit of the assay was 10(4) CFU g(-1) of feed. The amplification efficiency (Eff) of each PCR was determined using the LinRegPCR software and Eff differences between individual samples and standards were corrected for. When compared to plate counts, standard curve-1 slightly under-estimated the number of spores (mean=-2.47% of plate counts). A spore density-dependent Eff was found, and Eff for standard curve-1 could not be determined. Standard curve-2 over-estimated spore numbers when not corrected for individual Eff (mean=+5.46% of plate counts). Standard curve-2 Eff was independent (Eff(mean)=1.96) of spore density. The assay quantified the numbers of spores in feed samples very similar to plate counts (mean=+0.47% of plate counts), when standard curve-2 was used and individual Eff was accounted for.

A PCR test for identification of Haemophilus parasuis was optimized using the 16S rDNA sequences of the 15 serotype reference strains of H. parasuis. The test was evaluated on a collection of 218 Danish field isolates as well as on 81 representatives of 27 other species, including genetically affiliated species within Pasteurellaceae. In addition, DNA preparations from 56 H. parasuis isolates from North America were included. To obtain a test that was specific for H. parasuis, a multiplex PCR using 3 different primers was developed. The PCR test produced an amplicon of approximately 1090 bp only with representatives of H. parasuis. The test was further evaluated on 55 clinical samples from 16 Danish pigs suspected for being infected with H. parasuis, showing polyserositis or septicemia at autopsy as well as on 492 nasal swabs. The test was compared with the performance of a PCR test earlier published by Oliveira et al. [Oliveira, S., Galina, L., Pijoan, C., 2001. Development of a PCR test to diagnose Haemophilus parasuis infections. J. Vet. Diagn. Invest. 13, 495-501]. The sensitivity of the present PCR test was found to be slightly lower when applied on clinical samples from diseased pigs and 10-fold lower when tested on pure cultures of H. parasuis (5CFU and 0.5CFU/PCR reaction, respectively). Addition of 1.4 x 10(5) Escherichia coli to each PCR tube did not alter the sensitivity of the tests. No difference in sensitivity of the tests was observed when tested on purified DNA. On the other hand, the present PCR test was found to be 100% species specific for H. parasuis, in contrast to the PCR test of Oliveira et al., which also tested positive for strains belonging to A. indolicus, A. porcinus, and A. minor, species commonly occurring in the upper respiratory tract. However, when the PCR test of Oliveira et al. is used on samples from systemic locations the chances for false positive results are apparently low. The present PCR test represents a rapid and reliable method for genetically based identification of H. parasuis. The high species specificity of the test makes it suitable for detection of H. parasuis in clinical samples, regardless of the presence of affiliated species and contaminating flora. As the two PCR tests differ in sensitivity and specificity, the use of both PCR tests for different purposes is a possibility.

The microbial communities of the ileum and cecum of broiler chickens from a conventional and an organic farm were investigated using conventional culture techniques as well as cloning and sequencing of 16S rRNA genes. Eighty-five percent of the 557 cloned sequences were <97% related to known cultured species. The chicken ileum was dominated by lactobacilli, whereas the cecum harbored a more diverse microbial community. The cecum was dominated by a large group of bacteria with hitherto no close cultured relatives but most closely related to Faecalibacterium prausnitzii. Approximately 49 and 20% of the cecal clones belonged to this cluster in conventional and organic broiler chickens, respectively. We were, however, able to recover a number of these phylotypes by cultivation, and the isolates were shown to be butyric acid producers. The investigation was a descriptive rather than a comparative study of 2 different rearing systems; however, several differences were observed. For instance, Clostridium perfringens was found in significantly higher numbers in the birds from the organic farm compared with the conventional broilers, probably due to the addition of salinomycin to the conventional feed. In the ileum, the abundance of the different Lactobacillus species differed between the 2 broiler types. The culture-based and culture-independent techniques complemented each other well. Strengths and limitations of the different methods are discussed.

Terminal-restriction fragment length polymorphism (T-RFLP) was used to evaluate how to store intestinal specimens for bacterial community analysis. Bacterial communities are increasingly often described by means of DNA-based methods and it is common practice to store intestinal or faecal specimens either at -20 degrees C or -80 degrees C. In this study, samples of intestines from five different pigs were stored at -80 degrees C and -20 degrees C, respectively and a thawing and freezing procedure was carried out three times for each intestinal per pig per temperature. The cumulative sum of the T-RFLP peak heights (T-RF intensities) decreased as the temperature decreased. The composition of the bacterial community changed when stored at -80 degrees C compared to the samples stored at -20 degrees C. Thus it is recommended from this study that samples of intestinal content are stored at -20 degrees C before use for bacterial community analysis, instead of the current practice at -80 degrees C.

Abstract The impact of copper amendment on the bacterial community in agricultural soil was investigated by a 2-year field experiment complemented by short-term microcosm studies. In the field, the amendments led to total copper contents that were close to the safety limits laid down by European authorities. In parallel, bioavailable copper was determined with a copper-specific bioluminescent Pseudomonas reporter strain. The amounts of total Cu as well as of bioavailable Cu in the field declined throughout the experiment. Bacterial community structure was examined by terminal restriction fragment length polymorphism (T-RFLP) analysis of community DNA amplified with primers specific for 16S rDNA from the Bacteria domain, the Rhizobium-Agrobacterium group and the Cytophaga group. Similarity analysis of T-RFLP profiles from field samples demonstrated an impact of copper at the domain level and within the Rhizobium-Agrobacterium group. Comparable Cu effects were observed for microcosms, but in addition an impact on community structure within the Cytophaga group was observed.

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.

The survival of Brachyspira hyodysenteriae and Brachyspira pilosicoli was investigated at 10 degrees C in laboratory microcosms consisting of soil, porcine faeces, and in soil mixed with 10% porcine faeces, respectively. By plate spreading, survival of B. hyodysenteriae was found to be 10, 78 and 112 days in soil, soil mixed with 10% faeces, and in porcine faeces, respectively. The identities of the colonies on the plates were confirmed using PCR targeting 23S rDNA for specific detection of B. hyodysenteriae. A positive PCR signal could be obtained up to 112 days in all microcosms by direct extraction of DNA from microcosms followed by PCR. The survival time for B. pilosicoli was 119 days in pure soil and 210 days in soil mixed with 10% porcine faeces and in pure faeces, respectively, as determined by plate spreading followed by PCR. On the other hand, by direct extraction of DNA followed by specific detection by PCR. B. pilosicoli could be detected up to 330 days in all microcosms.Dot blot hybridisation with digoxigenin-labelled specific oligonucleotide probe targeting rDNA could not be used for direct detection of Brachyspira spp. from microcosms due to low sensitivity. However, it was used for confirmation of the identity of colonies and proved to be a useful technique. These results show that the two Brachyspira species may survive in outdoor environment for the times shown in these investigations using laboratory microcosms.

A feeding experiment was carried out over 42 d with four groups of broiler chickens fed experimental diets formulated to provide no supplementation, 20 mg zinc bacitracin, 60 mg salinomycin, or both feed additives in combination. During the fifth week of the experiment, four chickens from each pen were killed, and the contents of gizzard, duodenum, jejunum, ileum, ceca, and rectum were separately collected and pooled. In all intestinal segments, the pH and the concentration of lactic acid were measured, and the numbers of anaerobic bacteria, coliforms, lactic acid bacteria, lactobacilli, enterococci, and Clostridium perfringens were counted. In homogenates of pancreas obtained from four animals, the activities of amylase, lipase, trypsin, and chymotrypsin were measured. A significant growth-promoting effect was observed in the group receiving zinc bacitracin in combination with salinomycin. Zinc bacitracin significantly reduced the number of coliform bacteria in the ileum and increased the activities of amylase and lipase in pancreas homogenates. Supplementation with salinomycin and zinc bacitracin, alone or in combination, resulted in significantly lower counts of C. perfringens as well as Lactobacillus salivarius, which was a dominant lactic acid bacterium found in broiler intestinal contents. High numbers of these lactobacilli may play a role in broiler growth depression related to competition in nutrient uptake or impaired fat absorption due to bile acid deconjugation.

Bacterial communities in the large intestines of pigs were compared using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S ribosomal DNA. The pigs were fed different experimental diets based on either modified standard feed or cooked rice supplemented with dietary fibers. After feeding of the animals with the experimental diets for 2 weeks, differences in the bacterial community structure in the spiral colon were detected in the form of different profiles of terminal restriction fragments (T-RFs). Some of the T-RFs were universally distributed, i.e., they were found in all samples, while others varied in distribution and were related to specific diets. The reproducibility of the T-RFLP profiles between individual animals within the diet groups was high. In the control group, the profiles remained unchanged throughout the experiment and were similar between two independent but identical experiments. When the animals were experimentally infected with Brachyspira hyodysenteriae, causing swine dysentery, many of the T-RFs fluctuated, suggesting a destabilization of the microbial community.

Two groups of six 8-week-old pigs were challenged with 1x10(9) cfu Brachyspira (Serpulina) pilosicoli or Serpulina intermedia daily for 3 consecutive days to study the pathology of porcine colonic spirochetosis by scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with oligonucleotide probes targeting ribosomal RNA specific for B. pilosicoli and the genus Brachyspira/Serpulina. Six pigs served as noninoculated controls. The animals were euthanatized successively between postinoculation days 14 and 24. B. pilosicoli was reisolated in feces from all of the inoculated pigs; however, only two pigs developed transient watery diarrhea. S. intermedia was reisolated from four of the inoculated pigs, but clinical signs were not observed. Gross examination of the B. pilosicoli-infected pigs revealed dilated large intestines with a hyperemic mucosa, whereas the large intestines of the S. intermedia-inoculated pigs and the control pigs appeared normal. SEM examination of B. pilosicoli-infected pigs revealed degenerated epithelial cells and spirochetal colonization of the colonic mucosa in four pigs. By FISH, B. pilosicoli cells were found colonizing and invading the surface epithelium and the crypts in all the pigs. Spirochetal crypt colonization markedly exceeded the occurrence of spirochetes on the mucosal surface. SEM examination of S. intermedia-inoculated pigs revealed no abnormalities, and Serpulina cells were detected only sporadically in the otherwise normal-appearing mucosa of four pigs by FISH. The results provide further evidence that B. pilosicoli is associated with colitis in pigs, although the gross lesions are mild. The spirochete is capable of colonizing the large intestine, inducing mucosal damage, invasion of the crypt and surface epithelium, and focal infiltration of the lamina propria. In addition, the study shows the applicability of FISH for specific identification of B. pilosicoli in formalin-fixed tissue.

The localization of Serpulina hyodysenteriae in experimental swine dysentery was studied by fluorescent in situ hybridization (FISH) using an oligonucleotide probe targeting the 23S rRNA of S. hyodysenteriae. Nine 8-week-old pigs were challenged. Seven of the pigs were intragastrically dosed with 1 x 10(9) cfu S. hyodysenteriae for 3 consecutive days, whereas two pigs were infected by contact. Six non-challenged pigs served as negative controls. The challenged pigs developed clinical swine dysentery from 8 to 14 days postinfection with typical gross lesions. By FISH S. hyodysenteriae cells in huge numbers were found colonizing the mucus layer, the luminal epithelium and the crypts of the large intestinal mucosa. The orientation of the spirochaetes to the epithelium appeared to be random. Spirochaetes in clusters or as single cells were invading the epithelium and were occasionally detected in the adjacent lamina propria. The distribution of spirochaetes in the mucosa provides further evidence that S. hyodysenteriae is intimately associated with the mucus layer and the epithelium in a random pattern. Furthermore, the results demonstrate the applicability of FISH for specific detection of S. hyodysenteriae cells in clusters or as single cells in formalin-fixed tissue samples.

A fluorescent-labelledin situ hybridization method targeting rRNA was devised to facilitate specific identification and diagnosis of diarrhoea and colitis in pigs caused by the genus Serpulina, as well as to distinguish the species Serpulina hyodysenteriae and Serpulina pilosicoli in formalin-fixed colon tissue sections. A genus-specific oligonucleotide probe SER1410 targeting the five species of porcine Serpulina was thus designed. Furthermore, species specific oligonucleotide probes (Hyo1210, Pilosi209 and Pilosi1405) were also designed to detect, identify and differentiate S. hyodysenteriae and S. pilosicoli. These probes clearly demonstrated and possessed the desired specificity, when evaluated by whole cell hybridization on five reference strains and 20 isolates covering the five species of porcine Serpulina. Furthermore, the oligonucleotide probes were specific when used both, for the detection of Serpulina isolates at genus level as well as for specific detection of S. hyodysenteriae and S. pilosicoli in formalin-fixed colon tissue sections from pigs suffering from swine dysentery and porcine colonic spirochaetosis, respectively. Tissue sections were also used from pigs without any intestinal disorders as controls for estimating the specificity of the probes. The probes developed in this study thus had the potential of specific identification and histological recognition obtained in the formalin-fixed tissue samples., (Copyright 1998 Academic Press)

A polymerase chain reaction (PCR) was optimized to detect Lawsonia intracellularis in faeces from naturally infected pigs. By combining a boiling procedure to extract DNA and a nested PCR procedure, a detection limit at 2 x 10(2) bacterial cells per gram of faeces was achieved. The optimized PCR was used together with conventional culture techniques to detect Serpulina hyodysenteriae, weakly beta-haemolytic intestinal spirochaetes (WBHIS), Salmonella enterica, and haemolytic Escherichia coli, in a case control study to examine selected risk factors for the development of diarrhoea in growing pigs. Herds with diarrhoea were selected as cases and randomly chosen herds without diarrhoea were chosen as controls. Infection with L. intracellularis significantly enhanced the chance of diarrhoea. S. hyodysenteriae, WBHIS group IV (Serpulina pilosicoli), and S. enterica were isolated only from case herds which indicate that these species may influence the development of diarrhoea. In addition, herd-type had a significant impact, that is specific pathogen-free herds showed an odds ratio at 0.2 relative to conventional herds for the development of diarrhoea.

Fluorescent in situ hybridization targeting 16S ribosomal RNA was used for specific detection of the obligate intracellular bacterium Lawsonia intracellularis in enterocytes from pigs affected by proliferative enteropathy. A specific oligonucleotide probe was designed and the specificity of the probe was determined by simultaneous comparison with indirect immunofluorescence assay for detection of L. intracellularis in formalin-fixed tissue samples from 15 pigs affected by porcine proliferative enteropathy. We used 10 tissue samples from pigs without proliferative mucosal changes as negative controls. The results showed that the oligonucleotide probe is specific for L. intracellularis and that fluorescent in situ hybridization targeting ribosomal RNA is a suitable and fast method for specific detection and histological recognition of L. intracellularis in formalin-fixed tissue.

A 2470-bp section of the 23S ribosomal DNA from Serpulina hyodysenteriae and five biochemically different groups of weakly beta-haemolytic porcine intestinal Serpulina strains was sequenced. The similarity between the sequenced strains was high (96.85% to 99.84%). A phylogenetic tree was estimated by the maximum likelihood method. The sequenced strains formed three groups. Serpulina hyodysenteriae and biochemical group II ('S. intermedius') formed a cluster, but 20 nucleotide positions were different between the two, suggesting that biochemical group II is a separate species. Another cluster consisted of the closely related biochemical group IIIa ('S. murdochii') and IIIb/c (S. innocens) (99.84% similarity), while biochemical group IV (S. pilosicoli) constituted a separate group with a relatively low similarity (96.85% to 97.01%) to the other groups. Three primer pairs were designed for specific PCR detection of the clinically important S. hyodysenteriae and biochemical group II and IV. PCR amplification was accomplished with DNA extracted from bacterial colonies by a simple boiling procedure, and with DNA extracted directly from porcine stool samples using a bead beating extraction procedure. The level of detection for the direct extraction and amplification method was 5 x 10(5) cells added g-1 normal faeces.

Genetically engineered Pseudomonas sp. strain B13(FR1) was released into laboratory-scale marine ecosystem models (microcosms). Survival of the introduced population in the water column and the sediment was determined by plating on a selective medium and by quantitative competitive PCR. The activity of the released bacteria was determined by in situ hybridization of single cells with a specific rRNA-targeting oligonucleotide probe. Two microcosms were inoculated with 10(6) cells ml-1, while an uninoculated microcosm served as a control. The number of Pseudomonas sp. strain B13(FR1) cells decreased rapidly to ca. 10(2) cells ml-1 within 2 days after the release, which is indicative of grazing by protozoa. Three days after the introduction into seawater, cells were unculturable, but PCR continued to detect cells in low numbers. Immediately after the release, the ribosomal content of Pseudomonas sp. strain B13(FR1) corresponded to a generation time of 2 h. The growth rate decreased to less than 0.04 h-1 in 5 days and remained low, probably because of carbon limitation of the cells. Specific amendment of the microcosms with 10 mM 4-chlorobenzoate resulted in a rapid increase of the growth rate and an exponentially increasing number of cells detected by PCR, but not in resuscitation of the cells to a culturable state. The release of Pseudomonas sp. strain B13(FR1) into the microcosms seemed to affect only the indigenous bacterioplankton community transiently. Effects on the community were also apparent from the handling of water during filling of the microcosms and the amendment with 4-chlorobenzoate.

Microcosms were designed to facilitate studies of the fate, functioning, and ecological effects of microorganisms released into the aquatic environment. The microcosms were three-phase systems (sediment/water/air) with three compartments (a primary producer component, a herbivore grazer component, and intact sediment cores). The microcosms were validated by comparing gross ecological parameters and microbial community structure between the microcosms and the eutrophic Lake Bagsværd, which was simulated in the model. The photosynthetic potential and chlorophyll a concentrations were significantly lower in the microcosms than in the lake, which apparently was due to inorganic nutrient limitation. In the microcosms, total bacterial numbers and metabolic activity by [(3)H]thymidine incorporation were unaffected by the reduced algal biomass and primary production, simulating field conditions closely, with a strong dependence on temperature. Two days after filling the microcosms, the percentage of similarity of the microbial communities in the microcosm and Lake Bagsværd was 40%, measured by hybridizations of total microbial DNA. The similarity increased during the 10-day experimental period to 63-76%. In two experiments, Alcaligenes eutrophus AEO106(pRO101) was released into the microcosms. The release reduced the similarity between microcosms and lake to 2% and 27%, depending on the number of introduced cells. Concomitant to a decline in the A. eutrophus AEO106(pRO101) population, the similarity gradually recovered. It is concluded that the microcosms can simulate a freshwater lake ecosystem, but care has to be taken when extrapolating microcosm results to the source ecosystem because of the possible different selective conditions in the microcosm.