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PubMed1996

Rapid and efficient method to eliminate substances inhibitory to the polymerase chain reaction from animal fecal samples.

To detect pathogenic viruses in animal fecal specimens by polymerase chain reaction (PCR) assays, it is important to remove or inactivate PCR-inhibitory substances. Recently, it was reported that such inhibitory substances in human feces could be efficiently eliminated by a cationic surfactant, Catrimox-14 (Iowa Biotechnology, Iowa) during extraction of viral RNA. In the present report, Catrimox-14 was successfully applied to detect pathogenic viruses in fecal specimens from a variety of animals. By extraction of viral DNA in the presence of this cationic surfactant, the PCR assay could detect canine parvovirus (CPV) in all fecal specimens prepared from 13 kinds of animals, i.e., cat, chicken, cow, dog, gerbil, goat, golden hamster, horse, mouse, pig, rat, rabbit, or sheep. Pretreatment by gel-filtration or boiling failed to remove or inactivate the PCR-inhibitory substances in fecal specimens from mouse, goat, rat, and sheep.

Animals↗
PubMedDate not supplied

Influence of refrigeration and formalin on the floatability of Giardia duodenalis cysts.

Giardia duodenalis cysts obtained from fresh fecal samples, fecal samples kept under refrigeration and fecal samples treated with formalin were studied as to their floatability on sucrose solutions with the following specific gravities: 1,040 kg/m3; 1,050 kg/m3; 1, 060 kg/m3; 1,070 kg/m3; 1,080 kg/m3; 1,090 kg/m3; 1,100 kgm3; 1,150 kg/m3; 1,200 kg/m3; and 1,250 kg/m3, contained within counting-chambers 0.17 mm high. Cysts that floated on and those settled down as sediments were counted, and had their percentages estimated. Sucrose solutions of 1,200 kg/m3 specific gravity (the average specific gravity of diluting liquids employed in floatation techniques) caused to float 77.7%, 78.4% and 6.6% of the G. duodenalis cysts obtained, respectively, from fresh fecal samples, fecal samples kept under refrigeration, and fecal samples treated with formalin. Cysts obtained both from fresh fecal samples and fecal samples kept under refrigeration presented similar results concerning floatability. It was observed, however, that the treatment of feces with formalin diminished the cysts floatability under the various specific gravities studied. This results should influence, the recommendations for transport and storage of fecal samples used for parasitological coproscopy.

Animals↗
PubMed2001

Enzyme immunoassay of 17 beta-estradiol, estrone conjugates, and testosterone in urinary and fecal samples from male and female mice.

ELISA measures of 17 beta-estradiol, estrone conjugates, and testosterone were adapted for fecal and urinary samples from laboratory mice. We will report on validations of these assays and data from interacting males and females. Unconjugated gonadal steroids were consistently measurable in urine and feces of both males and females. Females that were parturient following insemination excreted relatively low levels of urinary testosterone compared to non-parturient females. The results are consistent with evidence that elevated androgens and estrogens are incompatible with intrauterine implantation of fertilized ova, and suggest that steroids in male urine could contribute to pheromonal action. These methods permit repeated noninvasive measurement of steroid activity in this species.

Animals↗
PubMed2006

Competitive metagenomic DNA hybridization identifies host-specific microbial genetic markers in cow fecal samples.

Several PCR methods have recently been developed to identify fecal contamination in surface waters. In all cases, researchers have relied on one gene or one microorganism for selection of host-specific markers. Here we describe the application of a genome fragment enrichment (GFE) method to identify host-specific genetic markers from fecal microbial community DNA. As a proof of concept, bovine fecal DNA was challenged against a porcine fecal DNA background to select for bovine-specific DNA sequences. Bioinformatic analyses of 380 bovine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode membrane-associated and secreted proteins. Oligonucleotide primers capable of annealing to select Bacteroidales-like bovine GFE sequences exhibited extremely high specificity (>99%) in PCR assays with total fecal DNAs from 279 different animal sources. These primers also demonstrated a broad distribution of corresponding genetic markers (81% positive) among 148 different bovine sources. These data demonstrate that direct metagenomic DNA analysis by the competitive solution hybridization approach described is an efficient method for identifying potentially useful fecal genetic markers and for characterizing differences between environmental microbial communities.

Animals↗
PubMed2006

Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR.

A multiplex real-time polymerase chain reaction (PCR) method was developed for the simultaneous detection of Enterocytozoon bieneusi (n = 30) and Encephalitozoon spp. (n = 3) in stool samples. The multiplex PCR also included an internal control to detect inhibition of the amplification by fecal constituents in the sample. The assay was performed on species-specific DNA controls (n = 22) and a range of well-defined stool samples (n = 140), and it achieved 100% specificity and sensitivity. The use of this assay in a diagnostic laboratory offers the possibility of introducing DNA detection as a feasible technique in the routine diagnosis of intestinal microsporidian infections.

Animals↗
PubMed2000

The detection of Coxiella burnetii from ovine genital swabs, milk and fecal samples by the use of a single touchdown polymerase chain reaction.

The polymerase chain reaction (PCR), targeting the repetitive transposon-like region of Coxiella burnetii (Trans-PCR), was evaluated for its ability to detect directly C. burnetii in genital swabs, milk and fecal specimens of ewes. By using a combination of centrifugation step, DNA purification using Qiamp Tissue kit followed by Trans-PCR assay, the efficiency for detection of coxiella in ewes milk samples was further improved and one C. burnetii-cell could be detected in 1ml of milk. In addition, an effective, simple and rapid method to remove PCR-inhibitory substances from fecal specimens by simply diluting the DNA template to 1:100 is described, which made the detection of one microorganism per mg of sample then possible. The results obtained from seropositive ewes proved that C. burnetii could also be detected in milk and fecal samples of naturally infected animals.

Animals↗
PubMed2003

Noninvasive reproductive steroid hormone estimates from fecal samples of captive female sea otters (Enhydra lutris).

Noninvasive methods were validated for monitoring fecal metabolites of reproductive hormones (total estrogens and progestagens) in sea otters. Several captive female sea otters were sampled an average of two to three times per week over at least 1 year to document the reproductive hormone levels within each major reproductive event (estrus, anestrus, luteal phase, and pregnancy). We report significant differences in reproductive hormone levels consistent with most reproductive categories, as well as the minimal number of consecutive samples needed to accurately determine pregnancy using these methods.

Analysis of Variance↗
PubMed1999

Establishment of the PCR system specific to Salmonella spp. and its application for the inspection of food and fecal samples.

We established the PCR detection system specific to Salmonella species using Salmonella enterotoxin gene (stn). The detection limit was one bacterial cell per one gram of fecal and minced-meat samples using enrichment procedure by Tripticase soy broth or Salmonella enrichment broth, respectively. We concluded that this PCR system is useful for the practical application in the field of the public hygiene.

Animals↗
PubMed2006

Identification of genotype 3 hepatitis E virus in fecal samples from a pig farm located in a Shanghai suburb.

Strains of hepatitis E virus (HEV) genotypes 1 and 4 have been detected on the Chinese mainland although there have been no previous reports of zoonotic genotype 3 HEV. In the present study, 65 swine fecal specimens were collected from five pig farms located in different Shanghai suburbs. RT-PCR and nested PCR were undertaken using partial nucleotide sequences of Open Reading Frame 2 (ORF2) of HEV to detect HEV RNA. Genetic analysis was based on alignments of an amplified 150-nt ORF2 sequence. RT-PCR revealed 15 HEV positive samples among 65-pig fecal specimens examined. Phylogenetic analysis of the amplified sequences indicated seven HEV strains belonged to genotype 3 and eight strains to genotype 4. This is the first time that genotype 3 hepatitis E virus has been identified on the Chinese mainland.

Animals↗
PubMed2004

Comparison of four different methods for Salmonella detection in fecal samples of porcine origin.

Performances of four detection methods were evaluated for recovery of Salmonella spp. in naturally contaminated fecal specimens of porcine origin. The NMKL 71 method consisted of enrichment in Rappaport-Vassiliadis broth and plating on xylose-lysine-desoxycholate medium, whereas the SP-VG-M002 method relied on a Diasalm enrichment followed by streaking on xylose-lysine-tergitol 4 agar (XLT-4). The VIDAS SLM method was composed of double enrichment in Muller-Kauffmann tetrathionate broth and in M broths before processing in a VIDAS device. If the results were positive, the VIDAS ICS immunoenrichment was performed and the result transferred onto three different selective media. The VIDAS ICS protocol is an immunoconcentration step followed by plating on XLT-4. Seventy-eight samples were tested with all four methods simultaneously, leading to 34 positive samples with at least one method. For this assay, VIDAS SLM revealed 31 positive samples (91.2%), whereas the average positive percentage of the three other methods was 37.3% (P < 0.001). Two-paired comparisons with the VIDAS SLM method were also performed. McNemar values were systematically highly significant (P < 0.001). The proportion of agreement was significantly inferior (P < 0.05) for the comparison of VIDAS ICS and VIDAS SLM (68.7%) compared with the two other paired comparisons (average percentage, 81.5%). The conclusion reached by this trial is that VIDAS SLM significantly improves the recovery of Salmonella in naturally contaminated fecal specimens. For the paired-comparisons, NMKL 71 and SP-VG-M002 were comparable in terms of efficiency, whereas the VIDAS ICS protocol, as established by the manufacturer for food samples only, seemed less efficient than the other two.

Animals↗
PubMed1990

[Thermophilic Campylobacter from fecal samples of animals and their behavior in the ganglioside (GM1)-ELISA, Vero cell test and salt aggregation test].

From 397 fecal specimens from apparently healthy and from diarrheic pigs, dogs, cats and cattle 59 strains (= 15%) of thermophilic Campylobacter (C.) spp. were isolated by culture. 39 strains were identified as C. coli and 18 as C. jejuni whereas 2 isolates could not be classified. None of the strains was found to be positive for cytotoxic enterotoxin in the GM1-ELISA. In the Vero-cell test 5 isolates showed a cytotoxic effect. The salt aggregation test (SAT) for indicating cell surface hydrophobicity was positive with 24 strains (5 C. jejuni, 19 C. coli). A correlation of isolation results with clinical manifestation could not be observed.

Animals↗
PubMed2005

Comparison of bacteroides-prevotella 16S rRNA genetic markers for fecal samples from different animal species.

To effectively manage surface and ground waters it is necessary to improve our ability to detect and identify sources of fecal contamination. We evaluated the use of the anaerobic bacterial group Bacteroides-Prevotella as a potential fecal indicator. Terminal restriction length polymorphism (T-RFLP) of the 16S rRNA genes from this group was used to determine differences in populations and to identify any unique populations in chickens, cows, deer, dogs, geese, horses, humans, pigs, and seagulls. The group appears to be a good potential fecal indicator in all groups tested except for avians. Cluster analysis of Bacteroides-Prevotella community T-RFLP profiles indicates that Bacteroides-Prevotella populations from samples of the same host species are much more similar to each other than to samples from different source species. We were unable to identify unique peaks that were exclusive to any source species; however, for most host species, at least one T-RFLP peak was identified to be more commonly found in that species, and a combination of peaks could be used to identify the source. T-RFLP profiles obtained from water spiked with known-source feces contained the expected diagnostic peaks from the source. These results indicate that the approach of identifying Bacteroides-Prevotella molecular markers associated with host species might be useful in identifying sources of fecal contamination in the environment.

Animals↗
PubMed1994

[Isolation of Cryptosporidium parvum oocysts from fecal samples--the combination of ether extraction and discontinuous sucrose gradients].

A calf and 50 mice were infected with Cryptosporidium parvum, and their fecal materials were collected and treated with ether extraction (EE), followed by discontinuous sucrose gradients (DSG) method. EE method was to remove some of fat or lipid from feces. Sediments were washed by centrifugation (1,500 x g for 10 min., 3 times) in phosphate-buffered saline and then these washed sediments were sieved sequentially through stainless steel screens with a final mesh of 250 (61 microns porosity) to remove other debris. After sieving, the materials were suspended in 2.5% potassium dichromate solution. Oocysts were counted by using a hemocytometer and the recovery rate of pure oocysts was calculated on the basis of the count. Following centrifugation (1,500 x g for 30 min.) by DSG method, most oocysts were recovered at the interface between a gravity of 1.103 and 1.064. The recovery rates of pure oocysts from the fecal suspension of the calf (3.8 x 10(7)/ml) and the mouse (3.2 x 10(6)/ml) treated with EE method were 81.6% and 51.6%, respectively. It is suggested that the recovery rate was dependent on the number of oocysts in each suspension treated with EE method. To get the 50% recovery rate, there must be more than 2 x 10(6) oocysts per ml of the fecal suspension treated with EE method. By the combination of the two methods it was possible to isolate C. parvum oocysts from normal feces of the calf and mouse as well as from diarrheic feces.

Animals↗
PubMed1992

[Evaluation of the immunoenzyme test (Elisa) in detecting Clostiridium difficile toxin A in fecal samples].

Currently, the method of choice in diagnosis of Clostridium difficile-associated intestinal diseases is the detection of toxin B in fecal specimens. This method is long (72 h) and can be realized in laboratories which have tissue culture facilities. Commercial agglutination test have been evaluated but they lack in specificity. An immunoenzymatic test has been recently commercialized for detection of toxin A. We have compared the results of this assay on 275 fecal specimens from patients suspected of having Clostridium difficile-associated intestinal diseases with the results obtained with the cytotoxicity test and the culture. Of the 275 fecal specimens, 58 were positive in cytotoxicity and 53 in Elisa. The overall sensitivity and specificity of the Elisa compared with cytotoxicity were 89.5% and 99.0% respectively. The immunoenzymatic test detecting Clostridium difficile toxin A is an easy test to perform in 2 h 15; it displays a good correlation with detection of toxin B and can be very useful in daily laboratory diagnosis.

Bacterial Toxins↗
PubMed2004

Detection of Campylobacter spp. in chicken fecal samples by real-time PCR.

A real-time PCR assay for detecting thermophilic Campylobacter spp. directly in chicken feces has been developed. DNA was isolated from fecal material by using magnetic beads followed by PCR with a prealiquoted PCR mixture, which had been stored at -18 degrees C. Campylobacter could be detected in less than 4 h, with a detection limit of 100 to 150 CFU/ml, in a fecal suspension. A bacterial internal control was added before DNA extraction to control both DNA isolation and the presence of PCR inhibitors in the samples. The assay was performed on 111 swab samples from a Danish surveillance program and compared to conventional culturing using selective enrichment. There was no statistically significant difference in performance between real-time PCR and culture by selective enrichment, and the diagnostic specificity was 0.96 with an agreement of 0.92. Therefore, the assay should be useful for screening poultry flocks for the presence of Campylobacter.

Animals↗
PubMed2006

Comparison of a brush-sampling fecal immunochemical test for hemoglobin with a sensitive guaiac-based fecal occult blood test in detection of colorectal neoplasia.

BACKGROUND: Fecal immunochemical tests (FIT) are an advanced fecal occult blood test (FOBT) technology that reduces barriers to population screening by simplifying the logistics of stool-sampling. The current study was conducted to undertake a paired comparison of a sensitive guaiac FOBT (GFOBT; Hemoccult II Sensa, Beckman Coulter, Fullerton, CA) with a brush-sampling FIT (InSure; Enterix, North Ryde, NSW, Australia), to determine whether this FIT improves detection of significant neoplasia. METHODS: Individuals sampled consecutive stools, at home, with both FIT and GFOBT sampling devices while following dietary restrictions appropriate for GFOBT. Study populations included a screening cohort (n = 2351) and a symptomatic diagnostic group (n = 161). Paired comparison of positivity rates was undertaken in those found to have cancer and/or significant adenoma (high-grade dysplasia, villous change, > or =10 mm, serrated histology or > or =3 polyps), benign pathology, or no pathology. RESULTS: Combined results for both cohorts showed that the FIT returned a true-positive result significantly more often in cancer (n = 24; 87.5% vs. 54.2%) and in significant adenomas (n = 61; 42.6% vs. 23.0%). Of all UICC Stage I cancers, the FIT was positive in 12 of 13 compared with 4 of 13 with the GFOBT (P = .002). In analyses of just the screening cohort, the FIT remained significantly better at detecting cancers and significant adenomas; the false-positive rate for any neoplasia was marginally higher with the FIT than the GFOBT (3.4% vs. 2.5%; 95% CI of difference, 0-1.8%), whereas positive predictive values were 41.9% and 40.4%, respectively. CONCLUSIONS: This brush-sampling FIT is more sensitive for cancers and significant adenomas than a sensitive GFOBT. As such, it should deliver greater reductions in colorectal cancer mortality and incidence than the GFOBT.

Adenoma↗
PubMed2002

Development and use of a simple polymerase chain reaction assay to screen for Helicobacter spp. and H. hepaticus in intestinal and fecal samples from laboratory mice.

A simple and sensitive duplex polymerase chain reaction (PCR) assay was developed for use in detection of Helicobacter species and H. hepaticus in laboratory mice. Bacteria were extracted and concentrated from fecal pellets and intestinal segments by use of buoyant density centrifugation. To improve quality assurance, an internal control (mimic) for detection of false-negative reactions was included. In addition, cartridges (Capillette) pre-filled with PCR reagents, were used to minimize the hands-on time required, thus reducing the risk of contamination with previously amplified material. Laboratory mice from Swedish animal houses sent to the National Veterinary Institute for health monitoring were found to have high prevalence of H. hepaticus.

Animal Husbandry↗
PubMed2004

New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples.

The application of a real-time quantitative PCR method (5' nuclease assay), based on the use of a probe labeled at its 5' end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 x 10(4) cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.

Adult↗