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PubMed2006

Quick and efficient purification of Giardia intestinalis cysts from fecal samples.

Giardia intestinalis is an intestinal parasite that has sparked considerable interest because of the public health problem it creates and because it is regarded as one of the earliest divergent eukaryotes. The present report describes a new method for quick, clean, and effective isolation of G. intestinalis cysts from fecal samples. The isolated cysts have the quality required for biochemical studies of the excystation process.

Animals↗

PubMed2005

Detection of multiple cephalosporin-resistant Escherichia coli from a cattle fecal sample in Great Britain.

We describe the isolation of multiple cephalosporin-resistant Escherichia coli from cattle feces collected from animals at slaughter in Great Britain. Six E. coli strains were isolated with distinct XbaI pulsed-field gel electrophoresis (PFGE) profiles and different mechanisms of cephalosporin resistance from a single fecal sample. Two of these strains were found to contain conjugative plasmids conferring resistance to extended-spectrum cephalosporins that were indistinguishable from each other by restriction endonuclease digestion. Sequence analysis of the plasmid-encoded ampC showed that they were identical to bla(CMY-2), previously described in multiple-drug-resistant Salmonella and E. coli from animals in other parts of the world. DNA sequence analysis of the chromosomal ampC promoter regions for three cephalosporin-resistant strains lacking CMY-2 was determined. Several mutations were detected in the isolates tested including changes at positions -42 and -32, which are known to increase promoter strength. This report represents the first isolation of E. coli containing bla(CMY-2) from cattle in Great Britain, and, also to our knowledge, the first demonstration of multiple cephalosporin-resistant strains in a single animal.

Animals↗

PubMed1995

[The occurrence of Listeria monocytogenes in fecal samples of pigeons].

Listeria spp. were isolated from faeces samples from 12 (3.4%) of 350 pigeons examined. Three isolates were identified as L. monocytogenes (1x serovar 1/2a, 1/2b, and 4ab), eight isolates were L. innocua (2x serovar 6a, 6x serovar 6b), and two isolates L. seeligeri (serovar 6b). One fecal sample yielded a mixed culture of L. monocytogenes and L. innocua. The results indicate that pigeons are not important in the epidemiology of listeriosis in man and animals.

Animals↗

PubMed1999

Decreased incidence of VanA-type vancomycin-resistant enterococci isolated from poultry meat and from fecal samples of humans in the community after discontinuation of avoparcin usage in animal husbandry.

The use of the glycopeptide antibiotic avoparcin (AVO) as a feed additive in animal husbandry of many European countries led in 1994-1995 to frequent isolation of VanA-type vancomycin-resistant enterococci (VRE) from commercially produced animal foodstuffs as well as from fecal samples of nonhospitalized persons in Germany (Saxony-Anhalt state). However, at the end of 1997, a decreasing number of such VRE was detected in frozen and fresh poultry meat (chickens and turkeys) from German producers. At this point in time, AVO had been discontinued in animal husbandry for more than 2 and one-half years in Denmark/Norway, nearly 2 years in Germany, and about 8-9 months in all countries of the European Community and Switzerland, respectively. VRE were then only detected in very low concentrations in one-quarter of the poultry meat samples (eight of 31, originating from 18 distinct German producers and bought in 12 different supermarkets). A decline of VRE prevalence was also observed in the gut flora of healthy persons (VRE carriers) in the same region (Saxony-Anhalt state, Germany), having fallen from 12% (12/100) in 1994 when AVO was being used to 6% (6/100) in 1996 and 3% (13/400) in 1997 after it was discontinued. These results likely indicate the importance of antibiotic selective pressure by glycopeptides such as AVO for the presence of VRE in animal meat products from commercial animal husbandry. Additionally, it underlines the role of animal products for the spread of resistant bacteria and transferable resistance genes to humans in the community.

Animal Husbandry↗

PubMed2026

Identification of aviadenovirus and dependoparvovirus in an Adélie penguin fecal sample from Cape Royds (Ross Island, Antarctica).

Adenoviruses (Adenoviridae) and their associated replication-deficient dependoparvoviruses (Parvoviridae) are recognized as significant components of the avian virome. While well-studied in domestic birds, these viruses remain underexplored in wild Antarctic birds. Information is limited on viruses circulating in penguins, particularly outside of the Antarctic Peninsula Region. Here we leverage a fecal sample collected for diet analyses to identify an aviadenovirus (37,731 nt) and two distinct dependoparvoviruses. The Adélie penguin (Pygoscelis adeliae) feces-derived adenovirus exhibits a genome organization consistent with members of the genus Aviadenovirus but shares only ~64% amino acid identity in the DNA polymerase protein with its closest known relative and thus represents a new species of aviadenovirus identified in penguins. Additionally, we identified two new dependoparvoviruses, one in length of 4,869 nt and a second of 4,162 nt. These two dependoparvoviruses are diverse, sharing <61% Rep amino acid identity with those of other dependoparvoviruses. Penguin dependoparvovirus 1 represents a new species. The co-detection of these viruses in a single seasonal sample could suggest a potential helper-satellite relationship in Adélie penguins. This study provides the first evidence of aviadenoviruses in Adélie penguin feces. These findings highlight the importance of surveillance to understand pathogen dynamics in isolated Antarctic populations.

Animals↗

PubMed2004

Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones.

In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science.

Adrenal Cortex↗

PubMed2006

A widely applicable protocol for DNA isolation from fecal samples.

Feces are increasingly used as sources of DNA for genetic and ecological research. This paper describes a new method for isolation of DNA from animal feces. This method combines multiple purification steps, including pretreatment with ethanol and TE, an inhibitor-absorber made of starch, the CTAB method, the phenol-chloroform extraction method, and the guanidinium thiocyanate-silica method. The new method is efficient according to PCR results of 585 fecal samples from 23 species and costs much less than the commercial kits. The protocol can be tailored to the specific purpose of examining different diets of animals and can be performed with routine laboratory reagents.

Animals↗

PubMed1997

The prevalence of shedding of Cryptosporidium and Giardia spp. based on a single fecal sample collection from each of 91 horses used for backcountry recreation.

Cryptosporidium parvum and Giardia duodenalis are now recognized as primary enteric pathogens in animals and humans. Regulatory agencies, such as the Environmental Protection Agency are under increasing pressure to reduce the concentration of these protozoa in surface waters. Given the popularity of recreational riding of horses on public land in California backcountry, concerns have been raised by various regulatory agencies as to whether horses used for backcountry recreation are a significant source of C. parvum and G. duodenalis for the environment. The prevalence of fecal shedding of Cryptosporidium and Giardia in horses with a history of being ridden in California backcountry during 1993 and 1994 was estimated. Using both direct fluorescent antibody and levitation centrifugation tests, none of 91 single-collection fecal samples from throughout California had Cryptosporidium oocysts or Giardia cysts. Horses ranged from 4 to 24 years of age. Because none of the 91 samples, collected 1 time from each horse, were positive and assuming that the sensitivity and specificity of the test methods employed were 100%, the highest probable prevalence of shedding for either protozoal pathogen was < 3.2% for the cohort of horses studied.

Age Factors↗

PubMed1998

Enzyme-linked immunosorbent assay based on recombinant human group C rotavirus inner capsid protein (VP6) To detect human group C rotaviruses in fecal samples.

A recent study showed that 43% of a population in the United Kingdom were seropositive for group C rotavirus. The higher than expected incidence may be due to limited diagnosis of acute human group C rotavirus infections because no routine test is available. Human group C rotavirus infections are routinely diagnosed by electron microscopy (EM) and a negative group A rotavirus enzyme-linked immunosorbent assay (ELISA) result. An antigen-detection ELISA was developed with hyperimmune antibodies raised to human group C rotavirus recombinant VP6 (Bristol strain) expressed in insect cells. The assay was used to screen fecal samples to determine the prevalence of group C rotavirus infection. Samples positive by ELISA were confirmed by EM, polyacrylamide gel electrophoresis of double-stranded RNA, or detection of the VP6 gene by reverse transcription-PCR. Retrospective analysis indicated a 1 to 2% detection rate of positivity among samples from patients with acute diarrhea.

Animals↗

PubMed2006

Detection of canine parvovirus in fecal samples using loop-mediated isothermal amplification.

Loop-mediated isothermal amplification (LAMP) is a novel, sensitive, and rapid technique for detection of genomic DNA. The end-product of the technique is a white precipitate of magnesium pyrophosphate that is visible without the use of gel electrophoresis. The LAMP method was applied to the detection of canine parvovirus (CPV) genomic DNA. A set of 4 primers, 2 outer and 2 inner, were designed from CPV genomic DNA targeting the VP2 gene. The optimal reaction time and temperature for LAMP were determined to be 60 minutes and 63 degrees C. On the basis of results for 50 canine fecal samples using polymerase chain reaction (PCR) analysis as the gold standard, the relative sensitivity of LAMP was 100% and the relative specificity was 76.9%. The detection limit of the LAMP method was 10(-1) median tissue culture infective doses (TCID50)/ml, compared with 10 TCID50/ml for PCR analysis. In addition to the advantage resulting from visual detection of the end product, the LAMP method is very rapid, requiring only 1 hour to complete. This assay would be a viable alterative to PCR analysis for diagnosis of CPV infection in dogs. The LAMP method holds promise for use as a diagnostic assay for CPV detection in a clinical setting.

Animals↗

PubMed1999

Direct quantification of the enteric bacterium Oxalobacter formigenes in human fecal samples by quantitative competitive-template PCR.

Homeostasis of oxalic acid appears to be regulated, in part, by the gut-associated bacterium Oxalobacter formigenes. The loss of this bacterium from the gut flora is associated with an increased susceptibility to hyperoxaluria, a condition which can lead to the formation of calcium oxalate crystalluria and kidney stones. In order to identify and quantify the presence of O. formigenes in clinical specimens, a quantitative-PCR-based assay system utilizing a competitive DNA template as an internal standard was developed. This quantitative competitive-template PCR test allows for the rapid, highly specific, and reproducible quantification of O. formigenes in fecal samples and provides a prototype for development of DNA-based quantitative assays for enteric bacteria.

Bacteria↗

PubMed1998

Genomic analysis of a pathogenicity island in uropathogenic Escherichia coli CFT073: distribution of homologous sequences among isolates from patients with pyelonephritis, cystitis, and Catheter-associated bacteriuria and from fecal samples.

Urinary tract infection is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far the most common etiologic agent. Uropathogenic strains have been shown to contain blocks of DNA termed pathogenicity islands (PAIs) which contribute to their virulence. We have defined one of these regions of DNA within the chromosome of a highly virulent E. coli strain, CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. The 57,988-bp stretch of DNA has characteristics which define PAIs, including a size greater than 30 kb, the presence of insertion sequences, distinct segmentation of K-12 and J96 origin, GC content (42.9%) different from that of total genomic DNA (50.8%), and the presence of virulence genes (hly and pap). Within this region, we have identified 44 open reading frames; of these 44, 10 are homologous to entries in the complete K-12 genome sequence, 4 are nearly identical to the sequences of E. coli J96 encoding the HlyA hemolysin, 11 encode P fimbriae, and 19 show no homology to J96 or K-12 entries. To determine whether sequences found within the junctions of the PAI of CFT073 were common to other uropathogenic strains of E. coli, 11 probes were isolated along the length of the PAI and were hybridized to dot blots of genomic DNA isolated from clinical isolates (67 from patients with acute pyelonephritis, 38 from patients with cystitis, 49 from patients with catheter-associated bacteriuria, and 27 from fecal samples). These sequences were found significantly more often in strains associated with the clinical syndromes of acute pyelonephritis (79%) and cystitis (82%) than in those associated with catheter-associated bacteriuria (58%) and in fecal strains (22%) (P < 0.001). From these regions, we have identified a putative iron transport system and genes other than hly and pap that may contribute to the virulent phenotype of uropathogenic E. coli strains.

Bacteriuria↗

PubMed2004

Enhanced retrieval of DNA from human fecal samples results in improved performance of colorectal cancer screening test.

Colorectal cancer accounts for more than 10% of all cancer deaths but is curable, if detected early. We reported previously on a stool-based screening test in which DNA from stool samples is subjected to genome analysis; sensitivity of the test has been limited in part by inefficiency of retrieving DNA from stool. Our aim was to test the impact of a new purification method that would increase the yield of human DNA from stool. DNA from 86 cancer and 100 non-cancer subjects (diagnosed by colonoscopy) were purified from stool with a new method for DNA recovery based on sequence-specific capture with acrylamide gel immobilized capture probes as well as with a previously developed magnetic bead-capture procedure. The new purification method gives an average 5.4-fold increase in the quantity of human DNA that can routinely be retrieved from fecal samples. The increased recovery of DNA corresponds with an increase in assay sensitivity from 53% (CI: 42 to 64%) to 70% (CI: 59 to 79%); P = 0.0005 (by McNemar's test), with no change in specificity. The newly developed sample preparation method mitigates a major problem in detecting rare cancer-associated genetic changes in heterogeneous clinical samples such as stool.

Acrylamide↗

PubMed2002

Quantification of uncultured Ruminococcus obeum-like bacteria in human fecal samples by fluorescent in situ hybridization and flow cytometry using 16S rRNA-targeted probes.

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.

Clostridium↗

PubMed2003

Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR.

A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42 degrees C for 1 h, at 99 degrees C for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup II, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and II, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples.

Adolescent↗

PubMed1978

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.

Animals↗

PubMed1995

A comparison of Eimeria oocysts isolated from litter and fecal samples from broiler houses at two farms with different management schemes during one growout.

Eimeria oocyst populations were monitored in broiler houses on two farms during a growout and prior to the placement of new chicks on the same litter. Numbers of oocysts in litter were enumerated at Weeks 0, 5, 6, 7, and 9 and in feces at Weeks 5,6, and 7 in broiler houses with different management practices (both farms had identical coccidiostat programs but Farm A had a history of poor performance whereas Farm B had excellent performance records). On Farm A the number of oocysts in litter increased (P < .05) at the time of slaughter (Week 7) and placement of a subsequent flock (Week 9) when compared with Day 0. The number of oocysts in feces on Farm A also increased (P < .05) from Week 5 to 7. On Farm B there were no differences in numbers of oocysts in litter or feces over time. Farm B had (P < .05) lower numbers of oocysts in litter than Farm A at Week 7. The number of oocysts in feces from Farm B were consistently lower (P < .05) than Farm A at Weeks 5, 6, and 7. There were no differences in the species of Eimeria in litter between farms; however, there were differences in the species composition over time. Small (Eimeria mitis) and medium (primarily Eimeria acervulina) oocysts increased significantly over time, whereas the large oocysts (Eimeria tenella and Eimeria brunetti) and Eimeria maxima did not change over time. An increase (P < .05) in large oocysts was observed in fecal samples from Farm A over time.

Analysis of Variance↗

PubMed2003

Noninvasive detection of new simian immunodeficiency virus lineages in captive sooty mangabeys: ability to amplify virion RNA from fecal samples correlates with viral load in plasma.

The sooty mangabey (SM) (Cercocebus atys) is the natural host of a simian immunodeficiency virus, termed SIVsm, which gave rise to human immunodeficiency virus type 2. Data on the geographic distribution, prevalence, and genetic diversity of SIVsm in the wild remains limited. To address this issue, noninvasive strategies based on screening SM fecal and urine specimens for SIVsm-specific antibodies and virion RNA (vRNA) were developed, and the results were correlated with viral loads in plasma. Twenty-three SIVsm-infected and 27 uninfected SMs were evaluated. Time-matched urine, fecal and plasma samples were collected over a 2-month period from 16 captive naturally infected SMs. The remaining 7 infected and 27 uninfected SMs were sampled once. Each specimen was subjected to enhanced chemiluminescence-Western blot analysis and nested reverse transcriptase (RT) PCR. The results showed that urine was highly sensitive (96%) and specific (100%) for detection of SIVsm antibodies, while fecal detection was much less sensitive (16%). Conversely, vRNA detection was more sensitive in feces (50%) than in urine (2%) samples. Fecal-vRNA detection correlated with viral loads in plasma (P < 0.002). SMs with detectable fecal vRNA had a mean viral load in plasma of 458,006 copies/ml, while those with undetectable fecal vRNA had a mean viral load in plasma of 29,428 copies/ml. Moreover, for every log increase in the viral load in plasma, the odds of detecting virus in fecal samples increased 87-fold. Genetic diversity of SIVsm in the SM colony was characterized by sequencing partial gag (846 bp) and gp43 (439 bp) fragments. Surprisingly, four new SIVsm lineages were identified, two of which were initially detected by fecal RT-PCR. This study documents the suitability of noninvasive methods for the detection and molecular characterization of new SIV variants. These assays will be useful for studying the phylogeny and epidemiology of SIVsm infections in the wild, and they hold promise as tools for investigating natural SIV infections in endangered nonhuman primates.

Animals↗