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PubMedDate not supplied

Experiments in DNA extraction and PCR amplification from bighorn sheep feces: the importance of DNA extraction method.

Reliability of genotyping is an issue for studies using non-invasive sources of DNA. We emphasize the importance of refining DNA extraction methods to maximize reliability and efficiency of genotyping for such DNA sources. We present a simple and general method to quantitatively compare genotyping reliability of various DNA extraction techniques and sample materials used. For bighorn sheep (Ovis canadensis) fecal samples we compare different fecal pellet materials, different amounts of fecal pellet material, and the effects of eliminating two DNA extraction steps for four microsatellite loci and four samples heterozygous at each locus. We evaluated 192 PCR outcomes for each treatment using indices of PCR success and peak height (signal strength) developed from analysis output of sequencer chromatograms. Outermost pellet material produced PCR results almost equivalent to DNA extracted from blood. Where any inner pellet material was used for DNA extraction, PCR results were poorer and inconsistent among samples. PCR success was not sensitive to amount of pellet material used until it was decreased to 15 mg from 60 mg. Our PCR index provides considerably more information relative to potential genotyping errors than simply comparing genotypes derived from paired fecal and blood or tissue samples. Our DNA extraction method probably has wide applicability to herbivores that produce pelleted feces where samples dry rapidly after deposition.

Animals↗
PubMed1982

Effect of unpalatable diets, food restriction and saccharin-adulterated diet on tryptic, chymotryptic, and amylolytic activity in pancreas, intestine and feces of rats.

Growing rats were fed either 10 to 17% protein (casein) diets whose taste was changed daily by addition of aversive stimuli. Under these circumstances food intake and body weight gain were reduced compared with controls fed unadulterated diets. More amylolytic activity per gram pancreas was found in rats fed a 10% protein, aversive diet and in their pair-fed controls fed restricted amounts of an unadulterated diet compared with ad libitum controls. More amylolytic activity per gram chyme of small intestine was also found in rats fed less than ad libitum amounts of a 10% protein diet. The chymotrypsin activity in the pancreas of rats fed a 17% protein, aversive diet was greater than in that of control animals or that of the pair-fed animals. The 2- to 10-fold higher proteolytic activity found in the small intestine and especially in the large intestine and feces of rats fed the aversive diet was specifically related to the adulteration of the diet with sodium saccharin. Feeding a diet containing sodium saccharin led to lower cecal pH. Under conditions of the present experiments, changes in digestive enzyme activity are not directly related to the aversive taste of the diet.

Amylases↗
PubMed2004

In vitro incubation of human feces with daidzein and antibiotics suggests interindividual differences in the bacteria responsible for equol production.

Daidzein can be metabolized to equol, dihydrodaidzein (DHD), and O-desmethylangolensin (ODMA) by intestinal bacteria. Only one third to one half of individuals produce equol, and evidence exists to support potential cancer-protective effects of equol production. We investigated the in vitro metabolism of daidzein by fecal bacteria and assessed the effect of several antibiotics on metabolism. Fresh or previously frozen feces from 7 equol producers and 6 nonproducers were incubated with daidzein, with or without antibiotics, for 5 d at 37 degrees C. With the exception of one previously frozen sample, fecal inoculates from equol producers converted daidzein to equol. Conversion occurred under anaerobic, but not aerobic conditions. Fecal inoculates from equol nonproducers did not produce equol, but some produced ODMA and DHD. Between-subject differences in the effects of antibiotics on daidzein metabolism were apparent. Some antibiotics inhibited the production of equol but had no effect on DHD production. These results suggest that several bacteria may be involved in daidzein metabolism, and that they may differ among subjects. This simple in vitro system can facilitate the study of factors influencing equol production and minimize the need for animal models or human interventions. Furthermore, these analyses can be conducted on fecal samples that have been frozen and stored.

Anaerobiosis↗
PubMed1994

Comparative aspects of steroid hormone metabolism and ovarian activity in felids, measured noninvasively in feces.

Noninvasive fecal assays were used to study steroid metabolism and ovarian activity in several felid species. Using the domestic cat (Felis catus) as model, the excretory products of injected [14C]estradiol (E2) and [14C]progesterone (P4) were determined. Within 2 days, 97.0 +/- 0.6% and 96.7 +/- 0.5% of recovered E2 and P4 radioactivity, respectively, was found in feces. E2 was excreted as unconjugated estradiol and estrone (40%) and as a non-enzyme-hydrolyzable conjugate (60%). P4 was excreted primarily as non-enzyme-hydrolyzable, conjugated metabolites (78%) and as unconjugated pregnenolone epimers. A simple method for extracting fecal steroid metabolites optimized extraction efficiencies of the E2 and P4 excretion products (90.1 +/- 0.8% and 87.2 +/- 1.4%, respectively). Analysis of HPLC fractions of extracted fecal samples from the radiolabel-injected domestic cats revealed that E2 immunoreactivity coincided primarily with the unconjugated metabolized [14C]E2 peak, whereas progestogen immunoreactivity coincided with a single conjugated epimer and multiple unconjugated pregnenolone epimers. After HPLC separation, similar immunoreactive E2 and P4 metabolite profiles were observed in the leopard cat (F. bengalensis), cheetah (Acinonyx jubatus), clouded leopard (Neofelis nebulosa), and snow leopard (Panthera uncia). Longitudinal analyses demonstrated that changes in fecal E2 and P4 metabolite concentrations reflected natural or artificially induced ovarian activity. For example, severalfold increases in E2 excretion were associated with overt estrus or exogenous gonadotropin treatment, and elevated fecal P4 metabolite concentrations occurred during pregnant and nonpregnant (pseudopregnant) luteal phases. Although overall concentrations were similar, the duration of elevated fecal P4 metabolites during pseudopregnancy was approximately half that observed during pregnancy. In summary, steroid metabolism mechanisms appear to be conserved among these physically diverse, taxonomically related species. Results indicate that this hormone-monitoring approach will be extremely useful for elucidating the hormonal regulatory mechanism associated with the reproductive cycle, pregnancy, and parturition of intractable and endangered felid species.

Animals↗
PubMed2004

Relative role of plutonium excretion with urine and feces from human body.

The ratio of plutonium content in 35 pairs of daily fecal and urine samples from 19 former MAYAK workers several decades after the end of occupational exposure was measured in clinical conditions. No dependence of the ratio Pu(feces)/Pu(urine) on plutonium aerosol transportability, sex, and age of workers was revealed in the late times after the end of occupational exposure. It was found that at the late times after the end of occupational exposure, the ratio of feces/urine is characterized by the lognormal distribution with the median value, 0.57, and error for this index characterized geometric deviation, sigmag = 1.12 Urinary and fecal excretions were analyzed after chronic exposure to inhaled plutonium compounds of different transportability for another group of 345 workers. During 500-16,000 d after the started chronic inhalation, plutonium biokinetic model ("Doses-2000") used in Southern Ural Biophysics Institute (SUBI) and based on the ICRP Publication 66 overestimated the feces/urine ratio by an order of magnitude as compared with the observed values. It indicates a necessity for further improvement of the biokinetic model used in SUBI.

Administration, Inhalation↗
PubMed1991

Sialylated oligosaccharides in human milk and feces of preterm, full-term, and weaning infants.

The amount of free and glycosidically bound sialic acid was quantitated in the oligosaccharide fraction of breast milk from nine women in the 2nd-3rd week of lactation. These amounts showed a certain individual variation but the amount of bound sialic acid was higher than the free sialic acid in each sample. A similar study on the feces from preterm and full-term breast-fed infants revealed that the amount of free sialic acid increased while the bound sialic acid decreased during maturation, which could possibly be a result of increasing activity of an intestinal sialidase in the newborn child. The fecal oligosaccharide patterns in one blood group A secretor breast-fed infant were studied every 2 months during weaning until the age of 1 year. It was seen that the fecal oligosaccharide pattern disappears, along with the blood group A-active compounds, with a corresponding decrease in the amount of breast milk in the diet.

Breast Feeding↗
PubMed1994

Detection of large fragments of the human milk mucin MUC-1 in feces of breast-fed infants.

Evidence is growing that high-molecular-weight glycoproteins (mucins) of human milk bind to certain pathogenic microorganisms and thus could interfere with their colonization of the infant gut. This investigation sought to determine whether one of the two principal milk mucins, MUC-1 (also known as PAS-0 and episialin), persists into the feces. Fecal samples from seven breast-fed and seven formula-fed infants, aged from 20 days to 6 months, were analyzed. Glycoproteins in the specimens, solubilized with sodium dodecylsulfate, mercaptoethanol, and heat, were resolved by gel electrophoresis. The gels were blotted on nitrocellulose sheets that were immunostained with monoclonal antibodies specific for epitopes in the tandem repeat region of MUC-1. Mucin fragments of approximately 200,000 MW were detected in specimens from three of the seven breast-fed subjects; only one of them showed evidence of the intact mucin. Four breast-fed and all seven formula-fed babies were negative for the mucin in their stools. While these results provide evidence of a variable degree of breakdown, we speculate that MUC-1 resists degradation in the gut and thus may help bind host-detrimental microorganisms and inhibit their colonizing and infecting actions.

Animals↗
PubMedDate not supplied

Detection of Giardia lamblia antigen in the feces by counterimmunoelectrophoresis.

The presence of Giardia lamblia antigen in stools of symptomatic and asymptomatic giardiasis patients was studied by counterimmunoelectrophoresis using anti-G. lamblia (Portland strain I) trophozoite antibodies. This test could detect antigen in feces of 94% of giardiasis patients. However, it failed to differentiate between symptomatic and asymptomatic Giardia infection. There were no false-positive results in children with other diseases. Antigen in the stools disappeared within 1 to 2 weeks after eradication of infection with specific treatment. The overall sensitivity of this test in detecting G. lamblia antigen in stool was 94%, and the specificity was 95%.

Antigens, Protozoan↗
PubMed1990

Bifidobacterium gallicum sp. nov. isolated from human feces.

A bifidobacterial isolate from human feces was found to have very low genetic relatedness to any previously described species of the genus. This strain, which also contained a unique type of peptidoglycan, L-lysine-L-alanine-L-serine (A3 alpha), is considered to represent a new species, which is designated Bifidobacterium gallicum. Its description is presented. The type strain is strain DSM 20093. gallicum. Its description is presented. The type strain is strain DSM 20093.

Amino Acid Sequence↗
PubMed1997

Phenotypic and phylogenetic characterization of some Eubacterium-like isolates containing a novel type B wall murein from human feces: description of Holdemania filiformis gen. nov., sp. nov.

A group of Eubacterium-like strains (designated group S14), isolated from the feces of healthy people, was characterized by biochemical tests, fatty acid analysis, cell wall murein analysis, and 16S rDNA analysis. Our results indicate that group S14 is phylogenetically a member of the Clostridium subphylum of the gram-positive bacteria. Despite a phenotypic resemblance to the genus Eubacterium, group S14 was shown to be phylogenetically distantly related to the type species of the genus, Eubacterium limosum. Group S14 showed a specific phylogenetic association with Erysipelothrix rhusiopathiae. Group S14 resembled Erysipelothrix in possessing the uncommon type B cell wall murein. Structural analyses, however, revealed the presence of a previously unknown B1 delta (L-Ala)-D-Glu-Gly-L-Lys murein type. Based on a 16S rRNA sequence divergence of greater than 10% with E. rhusiopathiae and the presence of a unique murein type, a new genus, Holdemania, is proposed for group S14, with one species, Holdemania filiformis. Type strain of H. filiformis is ATCC 51649.

Cell Wall↗
PubMed1995

Isolation and characterization of bile acid 7-dehydroxylating bacteria from human feces.

Methods for isolation of fecal 7 alpha-dehydroxylating bacteria are presented. A total of 219 strains were isolated from feces of healthy humans, and their ability to 7-dehydroxylate cholic, chenodeoxycholic, and ursodeoxycholic acids were examined. Of all the isolates, 14 strains were found to be capable of eliminating the hydroxy group at C-7 alpha and/or C-7 beta. All the isolates were strictly anaerobic, Gram-positive rods. Thirteen isolates were non-sporeforming bacteria showing certain saccharolytic properties with the production of acid and gas from dextrose, and were catalase-positive but indole-, lecithinase-, urease- and oxidase-negative. Based on the data available at present, it was concluded that they could be regarded as members of the genus Eubacterium. One strain, however was identified as Clostridium sordellii. The isolated strains capable of 7 alpha-dehydroxylating cholic acid and chenodeoxycholic acid were also able to oxidize the hydroxy group at C-7 alpha. Nine strains (10, 12, 36S, M-2, M-17, M-18, Y-98, Y-1112, and Y-1113) of the 7 alpha-dehydroxylating bacteria were confirmed to have 7 beta-dehydroxylation ability, but five strains (O-51, O-52, O-71, O-72, and Y-67) could not transform ursodeoxycholic acid to lithocholic acid.

Adult↗
PubMed2000

Coprobacillus catenaformis gen. nov., sp. nov., a new genus and species isolated from human feces.

Three strains of Eubacterium-like isolates from human feces were characterized by biochemical tests and 16S rDNA analysis. The phenotypic characteristics of the three strains resembled those of the genus Collinsella transferred from the genus Eubacterium recently. However, Eubacterium-like strains were phylogenetically members of the Clostridium subphylum of gram-positive bacteria, and these showed a specific phylogenetic association with Clostridium ramosum and C. spiroforme. C. ramosum and C. spiroforme are gram-positive, anaerobic, spore-forming bacteria that belong to the genus Clostridium, and the G + C contents are 26.0 and 27.4 mol%, respectively. However, the three Eubacterium-like strains had G + C contents of 32.1 to 33.1 mol% and were non-spore-forming rods. Based on phenotypic characteristics, we can differentiate these species, and furthermore, a 16S rDNA sequence divergence of greater than 9% with a new related genus, Coprobacillus, is proposed for the three strains, with one species, Coprobacillus catenaformis. The type strain of C. catenaformis is JCM 10604T.

Base Composition↗
PubMed2000

Phylogenic and phenotypic characterization of some Eubacterium-like isolates from human feces: description of Solobacterium moorei Gen. Nov., Sp. Nov.

Three isolated strains from human feces were characterized by biochemical tests and 16S rDNA analysis. Phylogenetic analysis revealed that these isolated strains were members of the Clostridium subphylum of gram-positive bacteria. The phenotypic characters resembled those of the genus Eubacterium, but these strains were shown to be phylogenetically distant from the type species of the genus, Eubacterium limosum. The strains showed a specific phylogenetic association with Holdemania filiformis and Erysipelothrix rhusiopathiae. Based on a 16S rDNA sequence divergence of greater than 12% with H. filiformis and E. rhusiopathiae, a new genus, Solobacterium, is proposed for three strains, with one species, Solobacterium moorei. The type strain of Solobacterium moorei is JCM 10645T.

Bacteria, Anaerobic↗
PubMed1990

Study of virus excretion in feces of diarrheic and asymptomatic calves infected with rotavirus.

Infection of calves by rotavirus could lead to diarrhea or subclinical disease. Therein, kinetic of viral excretion from a group of asymptomatic calves, previously exposed to a virulent strain of rotavirus, is compared to that of a group the calves of which had diarrhea associated with rotavirus infection. As determined by an enzyme-linked immunosorbent assay (ELISA), duration of rotavirus shedding in feces and maximum yield of virus antigen were similar within the two groups of calves. Rotaviruses isolated from the two above groups of animals were antigenically related as shown by neutralization tests, and showed a similar RNA electrophoretic profile. In conclusion, it is likely that both asymptomatic and diarrheic calves infected by rotavirus are equally a major source of contamination to contact healthy calves.

Animals↗
PubMed1999

Design of cluster-specific 16S rDNA oligonucleotide probes to identify bacteria of the Bacteroides subgroup harbored in human feces.

To develop a new simple method for identification of bacteria in the Bacteroides subgroup isolated from human feces, we designed a panel of four 16S rDNA-targeted oligonucleotide probes specific for each cluster of the Bacteroides subgroup. The probes Bac and bacvul were targeted to the Bacteroides cluster, and the probes Pre and Por have their target regions characteristic to those of the Prevotella cluster and the Porphyromonas cluster, respectively. The probes presented in this work were constructed to be specific for reference strains in each cluster of the Bacteroides subgroup and were not cross-hybridized with other major intestinal bacteria. The use of combination of these four probes will faciliate the identification of the clusters of the Bacteroides subgroup harbored in the intestine as compared to biological and biochemical testings.

Bacteroides↗
PubMed2000

Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand.

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.

Animals↗
PubMed1997

[Rapid detection of Escherichia coli 0157:H7 in feces by latex agglutination and immunochromatographic assay].

The rapid detection method of Escherichia coli O157 in feces by using the latex agglutination test kit (Prolex; Pro Lab Diagnostics) and the immunochromatic assay kit (NOW EH. E. coli; Binax) was studied. 176 fecal samples obtained from 154 healthy men and 22 patients who had diarrhea by E. coli O157 were examined. Tellurite cefixim sorbitol MacConkey (TC-SMAC), Prolex and NOW were used for studies and also these were done after the enriched culture by tellurite cefixime vancomycin toriptic soy broth (TCV-TSB). In the direct inoculation, 5 (23%) of 22 samples, 7 (32%) and 9 (41%), and after the enriched culture, 7 (32%), 10 (46%) and 11 (50%) were positive by culture, Prolex and NOW, respectively. On the other hand, 154 samples from healthy men were all negative in the direct inoculation, but in the enriched culture, 7 of 40 (18%) were positive by NOW. These samples were negative by boiled (100 degrees C, 15 minutes). Our results indicated that Prolex and NOW were useful for accurate and rapid diagnosis of E. coli O157 enteritis, and more sensitivity results were obtained with the enrichment broth.

Chromatography↗
PubMed2000

[Molecular investigation of pathogenic factors of suspected-diarrheogenic Escherichia coli isolates from patients feces].

One hundred fifty-one O serotypable Escherichia coli strains which were assumed diarrheogenic E. coli among 2,240 strains of E. coli isolated from the in- and outpatients stools with or without gastrointestinal symptoms at Kyorin University Hospital from February 1994 to September 1996 were examined for the relationship between the possession of eight pathogenic factor-related genes and gastrointestinal symptoms of the patients using the polymerase chain reaction (PCR) for these strains. The rate of possession of pathogenic factor-related genes in the E. coli examined was 20.5% (31 strains) and gastrointestinal symptoms were found in all the patients with these strains except one. In the patients without gastrointestinal symptoms, E. coli isolates that possesses these genes was detected in only one case during 61 cases. The respective genes detected were eaeA and astA in each 14 strains, VT1 in 6, VT2 in 5, ST1b in 4, aggR in 3 and LT in 2, ST1a and invE gene was not detected. In particular, the O157 strains were found in 55.6% (5/9 strains) for these genes, and individual strains had VT1, VT2, eaeA and astA genes simultaneously. In contrast, none of these related genes was found in 9 strains of enteroinvasive serotype but enteropathogenic E. coli-related genes were found in 3 strains. The rate of possession of the genes related to enterotoxigenic E. coli, O159 which was most frequently isolated was low as 2.3% (1/43 strains, astA gene) and there were strains showing low correlation to the state of possession of the genes with the O serotype. Since the prevalence of the gastrointestinal symptoms is clearly high for the case which possesses the strain of which the pathogenic factor-related gene was detected, it was suggested that detection of pathogenic factor-related genes in E. coli isolates from feces using the PCR could be an effective means to decide whether the bacteria concerned was a causal bacteria or not in clinical practice.

Diarrhea↗