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At least 469 records · Page 26Linked to original sources
PubMed1982

Rapid isolation of Yersinia spp. from feces.

Direct plating or cold enrichment or both have been used to isolate Yersinia spp. from feces. Freeze-shock double enrichment and KOH treatment have been recommended for recovery of Yersinia enterocolitica from surface waters and food, respectively. These techniques were evaluated as alternatives for rapid recovery of Yersinia spp. from feces. Stool samples were homogenized in buffered saline and autoclaved. Escherichia coli. Klebsiella pneumoniae, and Pseudomonas aeruginosa were each added to the suspension at a final concentration of 1.5 x 10(6) colony-forming units per ml. Yersinia cells were then added to a final concentration of 1.5 x 10(3), 1.5 x 10(4), 1.5 x 10(5), or 1.5 x 10(6) colony-forming units per ml. A total of 21 strains of Y. enterocolitica, 2 of Yersinia kristensenii, and 1 each of Yersinia intermedia and Yersinia fredriksenii were tested. For freeze-shock double enrichment, seeded stool samples were frozen overnight (-70 degrees C), transferred successively to m-tetrathionate broth (6 h. 37 degrees C) and selenite broth (2 h 37 degrees C), and plated on MacConkey, salmonella-shigella, and cellobiose-arginine-lysine agars for quantitation. For KOH treatment, seeded stool samples were mixed with 0.5% KOH at a ratio of 1:2 for 2 min and plated as described above. E. coli, K. pneumoniae, and P. aeruginosa were virtually eliminated after either method was used. All Yersinia strains were recovered after KOH treatment even at the lowest initial concentration (1.5 x 10(3) colony-forming units per ml). However, after freeze-shock double enrichment, not all strains were retrievable, and those isolates which were recovered were grown only from samples containing the highest number of Yersinia strains (1.5 x 10(6) colony-forming units per ml). KOH treatment of stool samples seems to be a viable substitute for more protracted methods of recovering Yersinia spp.

Cold Temperature↗
PubMed1982

Shedding of rotavirus in feces of sows before and after farrowing.

The purpose of this study was to determine whether sows shed rotavirus near the time of farrowing. Twelve sows purchased from a common source and seropositive for rotavirus were housed in isolation in farrowing crates from 5 days before to 3 weeks after farrowing. Fecal samples were collected at 3- to 4-day intervals and examined for the presence of rotavirus by direct electron microscopy. Samples were also treated with pancreatin and inoculated onto monkey kidney cells. Rotaviral antigens were detected by a direct immunofluorescence technique, and selected positive cultures were examined by immunoelectron microscopy. Rotavirus was detected in the feces of 5 of 12 sows as early as 5 days before to 2 weeks after farrowing. Diarrhea related to rotavirus developed in 4 of 12 litters. Two of these four litters were farrowed by sows which shed rotavirus at 7 and 10 and 14 days after farrowing. The results of this study indicate that sows immune to rotavirus can shed virus in their feces at a time when piglets are particularly susceptible to infection and that adult swine are of primary importance in the epidemiology of rotavirus as initiators of infection.

Animals↗
PubMed1982

Potassium tartrate-glycerol as a density gradient substrate for separation of small, round viruses from human feces.

Cesium chloride density gradients are frequently used for virus concentration or purification in the preparation of human feces for examination by electron microscopy, Disruption of some of the fecal viruses occurs if they are pelleted from the density gradient in an additional concentration step. This report highlights an important limitation imposed by the use of cesium chloride as a density gradient substrate in attempting to recover small, round, virus-like particles from feces and suggests an alternative substrate which preserves virus morphology without the use of additional protective agents.

Adult↗
PubMed1990

Detection of Cryptosporidium parvum oocysts in bovine feces by monoclonal antibody capture enzyme-linked immunosorbent assay.

A monoclonal antibody enzyme-linked immunosorbent assay (ELISA) was developed to detect Cryptosporidium parvum oocysts in bovine feces. Fecal oocysts were concentrated by centrifugation through Formalin-ethyl acetate solution and captured with monoclonal antibody 18.280.2 reactive with C. parvum oocysts. Captured oocysts were detected with goat anti-oocyst serum, following the addition of a peroxidase conjugate of rabbit anti-goat immunoglobulin and O-phenylenediamine substrate. The assay was specific for Cryptosporidium sp. oocysts and did not detect oocysts of Eimeria auburnensis, Eimeria bovis, Eimeria ellipsoidalis, or Eimeria zuernii. Assay sensitivity allowed detection of 3 x 10(5) oocysts per ml of feces, compared with 1 x 10(6) oocysts per ml detected by examination of acid-fast-stained fecal smears and 1 x 10(3) oocysts per ml detected by indirect immunofluorescence. The capture ELISA was suitable for diagnostic analysis of bovine fecal samples and for assessment of oocyst shedding in experimentally infected calves. This assay may also prove useful for diagnostic assessment of humans in which cryptosporidiosis is suspected.

Animals↗
PubMed1991

Purification and characterization of Giardia lamblia antigens in the feces of Mongolian gerbils.

In a recent study, we identified Giardia lamblia 65- and 70-kDa antigens in the feces of infected Mongolian gerbils. The 65-kDa antigen was from a strain isolated from a human with symptoms of giardiasis, and the 70-kDa antigen was from a strain isolated from a human with no symptoms of giardiasis. In this study, we used preparative electrophoresis and electroelution techniques to purify these antigens to a degree which showed a single discrete protein band on silver-stained polyacrylamide gels. By enzyme-linked immunoelectrotransfer blot, common epitopes on the 65- and 70-kDa antigens were indicated by their cross-reactivity with rabbit anti-65-kDa and anti-70-kDa sera. By indirect immunofluorescence assay, the cysts and trophozoites of the two strains cross-reacted with these sera. Of seven lectins tested, only concanavalin A bound to the 70-kDa antigen, suggesting a glycoprotein, and it possessed a low isoelectric point as assessed by preparative isoelectric focusing. Molecular mass estimations of these antigens by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis were similar to the 65- and 70-kDa estimations obtained by native polyacrylamide gradient gel electrophoresis. Although the 65- and 70-kDa antigens proved to be resistant to 100 degrees C heat and stable in storage for up to 25 months at -20 degrees C, neither appeared to be the same as a fecal G. lamblia antigen with similar molecular mass found by other investigators. This suggests that variable G. lamblia antigens may be found in the feces of infected humans.

Animals↗
PubMed1995

Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR.

A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV.

Animals↗
PubMed1996

Comparative evaluation of several techniques for purification of Cryptosporidium parvum oocysts from rat feces.

Three methods of isolating Cryptosporidium parvum oocysts from rat feces were evaluated. Oocysts were initially isolated by sucrose density gradient centrifugation. They were further purified by passage through a glass bead column or a Percoll gradient or by dialysis. Although oocysts recovered from the glass bead column and by dialysis were relatively free of fecal debris, only oocysts recovered from the Percoll gradient were free of bacteria. Recovered oocysts retained their antigenicity and infectivity. The ability to effectively recover oocysts from rat feces suggests that the laboratory rat may be a convenient substitute for ruminants in the propagation and maintenance of C. parvum oocysts for in vitro and in vivo use.

Animals↗
PubMed1997

Detection of Cryptosporidium parvum DNA in formed human feces by a sensitive PCR-based assay including uracil-N-glycosylase inactivation.

We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452-bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil-N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,000 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.

Animals↗
PubMed1997

Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet.

Feces↗
PubMed2000

Helicobacter mesocricetorum sp. nov., A novel Helicobacter isolated from the feces of Syrian hamsters.

A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the feces of Syrian hamsters. The bacterium grew as a thin spreading film at 37 degrees C under microaerobic conditions, did not hydrolyze urea, was positive for catalase and alkaline phosphatase, reduced nitrate to nitrite, did not hydrolyze hippurate, and was sensitive to nalidixic acid but resistant to cephalothin. Sequence analysis of the 16S rRNA gene and biochemical and phenotypic criteria indicate that the novel bacterium is a helicobacter. The novel bacterium is most closely related to the recently described mouse enteric helicobacter, Helicobacter rodentium. This is the first urease-negative Helicobacter species with nonsheathed flagella isolated from feces of asymptomatic Syrian hamsters. We propose to name this novel helicobacter Helicobacter mesocricetorum. The type strain is MU 97-1514 (GenBank accession number AF072471).

Alkaline Phosphatase↗
PubMed2001

Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate.

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).

Amino Acid Sequence↗
PubMed2002

Identification of two phylogenetically related organisms from feces by PCR for detection of Salmonella spp.

Two previously reported PCR methods were evaluated to determine whether they are as sensitive and specific as conventional culture methods in detecting Salmonella spp. from feces. Bovine and equine feces were enriched overnight in brain heart infusion broth and assayed using PCR methods and primer sets described by other investigators. A total of 774 fecal specimens were tested using a primer set (invE-A primer set) that amplifies a region spanning the invasin E and A genes of Salmonella enterica serovar Typhimurium. A subset of these fecal specimens (306 of the 774 total) were tested using primers (hisJ primer set) that amplify a portion of the histidine transport J gene. The PCR required 24 h to obtain results, whereas it took 5 to 7 days to identify Salmonella spp. by culture. PCR detection of Salmonella spp. using the hisJ primers and the invE-A primers had a sensitivity of 93.3 and 80%, respectively, and a specificity of 85.6 and 98.6%, respectively, compared with bacterial culture. Amplification of 42 culture-negative fecal specimens (of 306 total specimens) generated a DNA fragment that corresponded to the molecular weight of the amplified hisJ gene. The hisJ-generated amplicons from six culture-negative and six culture-positive specimens were sequenced and analyzed using DNA sequence alignment and phylogenetic analysis software. A neighbor-joining dendrogram of the DNA sequences of both sets of hisJ amplicons revealed two distinct groups-one group of amplicons from culture-positive specimens identical to the hisJ gene of S. enterica serovar Typhimurium and a second group of amplicons from culture-negative specimens that were more closely related to hisJ of S. enterica serovar Typhimurium than to other hisJ sequences present in nucleotide databases.

Animals↗
PubMed2003

Evaluation of protocol using gene capture and PCR for detection of Helicobacter pylori DNA in feces.

The route of transmission of Helicobacter pylori, which is usually acquired in childhood and is one of the most common bacterial infections in humans, remains undetermined. Mapping the distribution of H. pylori genotypes within families could help to determine the routes of transmission and risk factors. Here we describe a noninvasive method for obtaining H. pylori DNA isolates from the feces of children. Children presenting with gastrointestinal symptoms at the Royal Hospital for Sick Children were tested for gastric H. pylori colonization by using the 13C-urea breath test (UBT) and were asked to provide fecal samples, which were tested for H. pylori by using the HpSA fecal antigen test. DNA was purified from fecal samples by using a novel method of gene capture with subsequent H. pylori PCR analysis. Fifteen UBT-positive and 15 UBT-negative children participated in the study. The positive and negative predictive values for the assay were 80 and 100%, respectively. Fecal DNA purification followed by H. pylori PCR analysis is an effective tool for harvesting H. pylori DNA isolates from the feces of children. This technique may be developed to allow the diagnosis and noninvasive genotyping of H. pylori in children and their families.

Antigens, Bacterial↗
PubMed2005

Comparison of two target genes for detection and genotyping of Giardia lamblia in human feces by PCR and PCR-restriction fragment length polymorphism.

A PCR assay targeting the tpi gene was developed to detect and to genotype Giardia lamblia in human feces. Our assay was specific and discriminated between G. lamblia assemblages A and B. G. lamblia cysts isolated from human feces were also analyzed with two previously described PCR-restriction fragment length polymorphism (RFLP) assays, which are based on the detection of tpi or gdh genes. These RFLP analyses distinguished groups I and II within assemblage A or groups III and IV within assemblage B. Among 26 fecal samples from patients with sporadic giardiasis diagnosed by hospital laboratories, the tpi gene was amplified from 25 (96%) with our PCR assay, whereas only 21 (81%) samples were positive when the gdh gene was targeted. Of the 25 positive samples, nine (36%) contained assemblage A and 16 (64%) contained assemblage B. Thus, RFLP analysis classified eight samples (32%) in assemblage A group II, eight (32%) in assemblage B group III, and five (20%) in assemblage B group IV. The group could not be specified for four samples. The tpi and gdh genes of G. lamblia assemblage B were amplified from 14 (93%) of 15 samples collected only from French soldiers coming back from the Ivory Coast. All of these contained assemblage B group III. The PCR method developed is sensitive, simple, and specific and shows that the tpi gene is well adapted for G. lamblia genotyping.

Adolescent↗
PubMed1978

Human rotavirus and its antibody: their coexistence in feces of infants.

Rotavirus and its antibody were detected by paper disk solid-phase radioimmunoassay or electron microscopy in feces of infants and young children with acute diarrhea. The fecal specimens in which rotavirus was detectable often contained a high titer of antibodies, which were shown by radioimmunoassay to belong mainly to the immunoglobulin G class. Rotavirus was rarely detected in the specimens containing immunoglobulin A antibodies. By dissociation tests carried out by radioimmunoassay, it was shown that the rotavirus particles in some specimens had the same antibody-binding capacity as did cultured simian rotavirus (SA-11), but antibodies in feces usually had low avidity, probably resulting from enzymatic digestion.

Antibodies, Viral↗
PubMed2001

Rapid detection of Brevibacillus formosus BN53-1 in chicken feces.

Here we describe a rapid method for detecting the hydrogen sulfide-decomposing bacterium Brevibacillus formosus BN53-1 in chicken feces. The method, which can be adapted to the specific detection of a variety of useful eubacteria, is based on blot hybridization and polymerase chain reaction (PCR), and makes use of the genus or species hypervariable region of eubacterial 16S rDNA. The approximate limit of detection under the conditions we tested was 1.0 x 10(3) cells in 10 mg of chicken feces.

Animals↗
PubMed1983

Determination of immunoreactive trypsin, pancreatic elastase and chymotrypsin in extracts of human feces and ileostomy drainage.

The total daily amount of extractable cationic trypsin, chymotrypsin, and pancreatic elastase 2 in feces and ileostomy fluids has been studied in normal individuals and healthy colectomized subjects. Quantitation was performed using immunological assays with polyethylene glycol as a fecal marker. The extractable amount of each of these enzymes in the feces of normal individuals was less than 1 mg/24 h. However, in fecal extracts from antibiotic-treated normal individuals a 100-fold increase in immunoreactive cationic trypsin was observed, while chymotrypsin and elastase 2 were only 2- to 3-fold higher. In extracts from ileostomy fluids cationic trypsin, elastase, and chymotrypsin all showed mean values in the order of 50-200 mg/24 h. The characterization of the immunoreactivity of pancreatic proteases showed no qualitative differences when measured in duodenal juice or fecal and ileostomy extracts.

Adult↗
PubMed1987

Similarities between IgE in human feces and a chymotrypsin-digest of an IgE myeloma protein.

Immunoglobulin E (IgE) in human feces was characterized by radioimmunoassays with different specificities of the second antibodies, and by immunoprecipitation techniques. The fecal IgE was compared with an intact IgE myeloma protein and with Fc'' fragments of the IgE molecules, obtained after prolonged chymotrypsin-digestion of the IgE myeloma protein. The experiments showed that both the fecal IgE and Fc'' fragments shared the D epsilon 1 antigenic determinants while the intact IgE had both D epsilon 1 and D epsilon 2 determinants. They also shared thermal stability at 56 degrees C as compared to intact IgE. Fecal IgE and Fc'' fragments both showed rapid anodal migration in an electric field, different to that of intact IgE. A tandem crossed immunoelectrophoresis technique provided signs of immunological identity between the precipitates of fecal IgE and those of the Fc'' fragments. It is therefore likely that most, if not all, IgE in human feces consists of Fc" fragments.

Child↗