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PubMed1997

A comparison of conventional microscopy, immunofluorescence microscopy and flow cytometry in the detection of Giardia lamblia cysts in beaver fecal samples.

A variety of domestic and wild animals are considered to be potential sources of giardiasis in humans. As a result, numerous studies have been reported on the prevalence of Giardia lamblia infection in animals. The majority of these surveys have involved various floatation techniques followed by conventional microscopy in order to detect cysts in fecal samples. Immunofluorescence microscopy has become popular in recent years for the detection of G. lamblia cysts in both clinical and environmental samples. This technique can be automated by combining it with flow cytometry. The present study represents a direct comparison of conventional microscopy, immunofluorescence microscopy, and flow cytometry in terms of their relative efficiency in the detection of G. lamblia cysts in beaver fecal samples. As a result of viewer fatigue, or low cyst concentrations, false negatives were common with conventional microscopy, leading to low prevalence estimates. By specifically targeting the cysts, immunofluorescence microscopy provided more reliable results in a shorter time than conventional methods. When flow cytometry was used in combination with immunofluorescence, a larger number of samples could be examined in a relatively short period of time. The results obtained indicated that this technique allowed for more consistent recognition than either conventional or immunofluorescence microscopy of positive samples containing smaller numbers of cysts.

Animals↗
PubMed1999

Demonstration of group C rotaviruses in fecal samples of diarrheic dogs in Germany.

Three out of 9 fecal samples from diarrheic dogs, which were positive for rotavirus by electron microscopy, revealed RNA-migration patterns identical to those of porcine group C rotavirus when studied by polyacrylamide gel electrophoresis (PAGE). The results were further confirmed by solid phase immuno electron microscopy (SPIEM). This is the first report of the occurrence of group C rotaviruses in dogs.

Animals↗
PubMed1980

Incompatibility of R plasmids derived from Salmonella and Escherichia coli strains isolated simultaneously from a bovine fecal sample.

Seven strains of Salmonella typhimurium were isolated from 2 fecal samples of calves, and the strains were resistant to ampicillin, chloramphenicol, kanamycin, nalidixic acid, streptomycin, sulfadimethoxine, and tetracycline. Also, 11 Escherichia coli strains were obtained from 1 of the 2 samples, and these strains possessed the identical drug resistance patterns as did the salmonellae. The thermosensitive R plasmids conferring sextuple drug resistance and mercury resistance from Salmonella strains were Fi- (no fertility inhibition) and belonged to incompatibility group H1, whereas conjugative 6 R plasmids isolated from 11 E coli strains were Fi+ (fertility inhibition) and belonged to incompatibility group FII. Thermosensitive R plasmids conferring sextuple drug (ampicillin, chloramphenicol, kanamycin, streptomycin, sulfadimethoxine, and tetracycline) have not been reported in Japan. Genetic relationship of R plasmids derived from the multiresistant strains of Salmonella and E coli was discussed on the basis of incompatibility properties of R plasmids.

Animals↗
PubMed2005

Stress hormones in mammals and birds: comparative aspects regarding metabolism, excretion, and noninvasive measurement in fecal samples.

A multitude of endocrine mechanisms are involved in coping with challenges. Front-line hormones to overcome stressful situations are glucocorticoids (GCs) and catecholamines (CAs). These hormones are usually determined in plasma samples as parameters of adrenal activity and thus of disturbance. GCs (and CAs) are extensively metabolized and excreted afterwards. Therefore, the concentration of GCs (or their metabolites) can be measured in various body fluids or excreta. Above all, fecal samples offer the advantages of easy collection and a feedback-free sampling procedure. However, large differences exist among species regarding the route and time course of excretion, as well as the types of metabolites formed. Based on information gained from radiometabolism studies (reviewed in this paper), we recently developed and successfully validated different enzyme immunoassays that enable the noninvasive measurement of groups of cortisol or corticosterone metabolites in animal feces. The determination of these metabolites in fecal samples can be used as a powerful tool to monitor GC production in various species of domestic, wildlife, and laboratory animals.

Adrenal Glands↗
PubMed2001

[Epidemiological study on vancomycin-resistant enterococci from fecal samples in the east area of Japan].

Recently, Vancomycin-resistant enterococci (VRE) have become important nosocomial pathogens in the world. In Japan, the VRE-infection was first reported in 1996. However, an epidemiological study on VRE has not been aggressively done in Japan. We conducted a survey study to explore the incidence and antimicrobial susceptibility of vancomycin-resistant enterococci isolated from fecal samples at 45 hospitals in the east area of Japan (Kanto, Koshinetsu, Tohoku, and Hokkaido) during June 1998 to March 1999. The Enterococcosel agar containing vanocomycin (BBL) was used for screening VRE from fecal samples in each hospital. The susceptibilities of the isolates to 8 antimicrobials were determined by the broth microdilution method and the definitions of resistance were based on current standards of the NCCLS standards. The VRE genotypes (vanA, vanB, vanC1, and van C2/3) were confirmed by amplifying the respective genes by PCR. Eight hundred and ninety four strains of enterococci were tested by the microtiter plates hybridization method (WAKUNAGA SEIYAKU, Japan). One thousand five hundred eighty three strains of enterococci were collected from 6,914 patients in 45 hospitals. These strains included 72 (4.5%) strains Enterococcus faecalis, 33 (2.1%) strains Enterococcus faecium, 17 (1.1%) strains Enterococcus avium, 1,040 (65.7%) strains Enterococcus gallinarum, 386 (24.4%) strains Enterococcus cassliflavus, and 35 (2.2%) strains Enterococcus flavescens. These strains of vancomycin-resistant E. faecalis were isolated from 3 patients, two of these 3 strains had van A gene and other one had van B gene. Those 3 strains were in the Kanto area, and 2 of 3 strains were in Tokyo, Generally, though van A type VRE was highly resistaant to both vancomycin and teicoplanin. In our study, two strains of van A type E. faecalis were highly resistant to vancomycin (MICs > 128 micrograms/ml) and susceptible to teicoplanin with MICs 4 micrograms/ml. Those two strains were different in susceptibilities of minocycline and ofloxacin. The result of the analysis of PFGE had also different patterns. VanB type E. fecalis was highly resistant to vancomycin and susceptible to teicoplanin (MICs 0.25 microgram/ml). For ampicillin and imipenem, 3 strains of E. faecalis were susceptible (MIC < or = 1 microgram/ml). One of 562 strains of E. gallinarum had vanB and vanC1 genes and was moderately resistant to vancomycin and susceptible to teicoplanin. All strains of E. casseliflavus and E. flavescens had vanC2/C3 gene only. All strains of E. faecium and E. avium did not detect van genes. From this result, it was supposed that VRE were very rare in the east of Japan.

Enterococcus↗
PubMed2001

Specific detection of Neospora caninum oocysts in fecal samples from experimentally-infected dogs using the polymerase chain reaction.

Neospora caninum oocysts, passed in the feces of a definitive host (dog), were isolated, and genomic DNA was extracted. A polymerase cahin reaction (PCR) targeting the N. caninum-specific Nc 5 genomic sequence was performed using the isolated DNA. A synthesized competitor molecule containing part of the Nc 5 sequence was included in the assay as a check against false-negative PCR results and to quantify N. caninum oocyst DNA in fecal samples. A standard curve of the ratio of fluorescence intensity of PCR-amplified competitor to that of oocyst DNA was constructed to compare oocyst equivalents from fecal samples containing unknown numbers of N. caninum oocysts and to assess the sensitivity of the assay. The specificity of the assay was determined using the Nc 5-specific primers in PCR assays against other parasites likely to be found in canine feces. Genomic DNA sequences from the canine coccidians Hammondia heydorni, Cryptosporidium parvum, Sarcocystis cruzi, S. tenella, and Isospora ohioensis and the canine helminth parasites Strongyloides stercoralis, Toxocara canis, Dipylidium caninum, and Ancylostoma caninum were not amplified. In addition, genomic DNA sequences from oocysts of coccidian parasites that might contaminate dog feces, such as Hammondia hammondi, Toxoplasma gondii, or Eimeria tenella, were not amplified in the PCR assay. The assay should be useful in epidemiological surveys of both domestic and wild canine hosts and in investigations of oocyst biology in experimental infections.

Animals↗
PubMed1983

[Megasphaera elsdenii--occurrence in 2,255 fecal samples of humans, chimpanzees and mice].

In human gnotobiotic studies the number of cfu/g feces expresses the interspecies-specific relations within this biotop. In our study we tried to culture the resident anaerobe microflora diluting 1 g feces to 10(-6) - 10(-9), and using the cystein-blood-agar (Beerens mod. Werner) together with the dextrose-water-blue agar. M. elsdenii appear as big, smooth and yellow colonies, which may be recognised at "first sight" diagnosis. In a retrospective study of 2255 fecal samples we compared the isolation-rate of M. elsdenii in the range of greater than or equal to 10(7)/g feces from healthy people to that of persons with gastrointestinal troubles. M. elsdenii has not been isolated from fecal samples of healthy people but in 12% from those of adults and 7.4% from children, both suffering from gastrointestinal disorders. So we consider that M. elsdenii in the range of greater than or equal to 10(7)/g feces does not belong to the resident anaerobe flora of the intestine. Further studies have to establish wether its normal range may be 10(3)-10(4)/g feces like that of C. perfringens. Comparing the isolation-rate from patients with colon-carcinoma to that of patients with dermatological affections, we found a relation of 8% to 20%. The biochemical activities of our isolates were according to the Anaerobe Laboratory Manual. Additional tests e.g. API-Zym and others make us assume an activity inner the metabolism of fatty-acids and amino acids e.g. inner the aspartat and pyruvat-family.

Adult↗
PubMed1996

PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples.

PCR procedures based on 16S rRNA gene sequences specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human (adult and baby) feces and animal (rat, mouse, cat, dog, monkey, and rabbit) feces. Fusobacterium prausnitzii, Peptostreptococcus productus, and Clostridium clostridiiforme had high PCR titers (the maximum dilutions for positive PCR results ranged from 10(-3) to 10(-8)) in all of the human and animal fecal samples tested. Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Eubacterium limosum also showed higher PCR titers (10(-2) to 10(-6)) in adult human feces. The other bacteria tested, including Escherichia coli, Bifidobacterium adolescentis, Bifidobacterium longum, Lactobacillus acidophilus, Eubacterium biforme, and Bacteroides distasonis, were either at low PCR titers (less than 10(-2)) or not detected by PCR. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

Adult↗
PubMed2006

Identification of Pentatrichomonas hominis in feline fecal samples by polymerase chain reaction assay.

Pentatrichomonas hominis is considered to be a commensal protozoan of the vertebrate digestive tract. On the basis of light microscopic examination of feces, some investigators presumptively identified P. hominis as a causative agent of feline diarrhea. However, molecular identification of P. hominis infection in the cat has not been reported. Another trichomonad, Tritrichomonas foetus, is recognized as an intestinal pathogen in cats and often presumptively diagnosed on the basis of the presence of trichomonads in diarrheic feces. It is of importance to determine if cats are natural hosts for P. hominis, as the presence of this organism could result in inaccurate assumption of T. foetus infection. In this study, we used a species-specific PCR assay to identify P. hominis 18S rRNA genes in fecal samples collected from a convenience population of cats in which a high prevalence of T. foetus infection had been previously identified (cat show) or suspected (submitted for T. foetus diagnostic testing). The prevalence of T. foetus infection in these samples was 31% and 28.6%, respectively. P. hominis infection was identified by PCR of DNA extracted from feces of five cats (1.9% and 2.1% of fecal samples, respectively). All cats in which P. hominis was identified were also infected with T. foetus. PCR identification of P. hominis infection in the cat should facilitate future studies to determine the pathogenicity of this species and enable differentiation of P. hominis from other known or as-yet unidentified species of trichomonads that may infect cats.

Animals↗
PubMed2005

Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. Increasing evidence indicates that hepatitis E is a zoonotic disease. There exist four major genotypes of HEV, and HEV isolates identified in samples from pigs belong to either genotype 3 or 4. Genotype 1 and 2 HEVs are found exclusively in humans. To determine whether genotype 1 and 2 HEVs also exist in pigs, a universal reverse transcription-PCR assay that is capable of detecting all four HEV genotypes was used to test for the presence of HEV RNA in serum and/or fecal samples from pigs in Thailand, where genotype 1 human HEV is prevalent, and from pigs in Mexico, where genotype 2 human HEV was epidemic. In Thailand, swine HEV RNA was detected in sera from 10/26 pigs of 2 to 4 months of age but not in sera from 50 pigs of other ages. In Mexico, swine HEV RNA was detected in 8/125 sera and 28/92 fecal samples from 2- to 4-month-old pigs. Antibodies to swine HEV were also detected in about 81% of the Mexican pigs. A total of 44 swine HEV isolates were sequenced for the open reading frame 2 gene region. Sequence analyses revealed that all swine HEV isolates identified in samples from pigs in Thailand and Mexico belong to genotype 3. Phylogenetic analyses revealed that minor branches associated with geographic origin exist among the swine HEV isolates. The results indicated that genotype 1 or 2 swine HEV does not exist in pigs from countries where the respective human HEV genotype 1 or 2 is prevalent. It is likely that only genotype 3 and 4 HEV strains have zoonotic potential.

Animals↗
PubMed2003

Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine.

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx(1), and stx(2) genes and isolates harboring the eaeA, stx(1), and hly(933) genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.

Animals↗
PubMed2006

Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig.

All feces produced during 24 h were collected from five pigs and cortisol and immunoreactive cortisol metabolites (CICM), and IgA were quantified. Within pigs, the concentrations of CICM and IgA varied extensively between random samples obtained from a single fecal dropping, and deviated in most cases significantly from the true concentration measured in total fecal output (CV 6.7-130%). The CICM and IgA contents varied considerably (CV 8.1-114%) within and between individual fecal droppings from the same pig compared to the total fecal excretion. In conclusion, single random samples could not be used to reliably quantify the total fecal concentration or excretion of CICM or IgA in pigs. Analyses of all feces collected during shorter periods than 24 h did not provide an accurate estimate of the daily excretion of CICM. Thus, the concentration of stress sensitive molecules in random single fecal samples as an indicator of animal welfare should be interpreted with prudence.

Animals↗
PubMed2005

Development of a nested PCR method targeting a unique multicopy element, ISMap02, for detection of Mycobacterium avium subsp. paratuberculosis in fecal samples.

This study describes the development of a nested PCR assay that uses a unique element (ISMap02) for Mycobacterium avium subsp. paratuberculosis that is present at six copies within the genome. In addition, the sensitivity of the assay with this element was compared to the sensitivity of detection of the IS900 element in both conventional and real-time PCR assays. The specificity of the ISMap02 element was evaluated by PCR of the DNA extracted from isolates of M. avium subsp. paratuberculosis and M. avium subsp. avium, as well as DNA from M. fortuitum, M. scofulaceum, M. phlei, M. smegmatis, and M. gordonae. Only M. avium subsp. paratuberculosis DNA was detectable after amplification with the ISMap02 primers. The sensitivity of detection for the ISMap02 element in either a conventional or a real-time PCR format was less than 100 fg DNA or 10(2) CFU/ml in serial titration curves with pure bacteria. These results were comparable to those obtained for the IS900 element. Experimental spiking of a negative fecal sample followed by M. avium subsp. paratuberculosis DNA extraction resulted in detection thresholds of 10(2) CFU/g for the IS900 element and 10(3) CFU/g for the ISMap02 element by using a real-time PCR format, but this sensitivity dropped 10-fold for both elements in a conventional PCR format. Analyses of fecal samples obtained from naturally infected animals demonstrated a sensitivity for the detection of M. avium subsp. paratuberculosis DNA by use of the ISMap02 element similar to that achieved by use of the IS900 element when it was used in a conventional PCR format. The real-time PCR format improved the levels of detection of both elements, but not to a significant degree. In conclusion, the ISMap02 element provides a very sensitive and specific alternative as a diagnostic reagent for use in PCR assays for the detection of paratuberculosis.

Animals↗
PubMed2001

Occurrence of Shiga toxin-producing Escherichia coli in fecal samples from children with diarrhea and from healthy zebu cattle in Uganda.

Fecal samples collected from 237 diarrheic infants in Kampala, Uganda and from 159 healthy cattle from a ranch in the Central Region of Uganda were investigated for the presence of Escherichia coli O157 and other types of Shiga toxin-producing E. coli (STEC). E. coli O157 were not detected in 150 stool samples from children which were cultivated on sorbitol MacConkey agar. A search for all types of STEC performed on 87 additional human stool samples with an enzyme-immunoassay for Shiga toxins (Premier EHEC) was also negative. Forty-two stool samples from infants were additionally investigated for enteropathogenic E. coli (EPEC) by DNA-hybridization with an eae-specific gene probe. Compared to STEC, EPEC were frequent and found in six (14.3%) of these 42 randomly selected stool specimens. We were further interested in the role of cattle as a reservoir for STEC in Uganda. STEC were isolated from 45 of 159 cattle from a herd in the Central Region of Uganda. STEC strains from cattle belonged to 16 different O- and nine different H-types and nine O:H types were identical to those found in bovine STEC from other continents. Only one bovine STEC strain was positive for the eae-gene, and O-groups associated with enterohemorrhagic E. coli (EHEC) types (O26, O103, O111, O145 and O157) were not found. Our report demonstrates that STEC are not frequent in urban children in Uganda, but domestic cattle were identified as an important natural reservoir for these organisms in this country.

Animals↗
PubMed2005

Performance of an immunoenzymatic assay for Cryptosporidium diagnosis of fecal samples.

We evaluated the diagnostic performance of a Cryptosporidium immunoenzymatic assay (ELISA). Fecal samples were collected from 94 HIV-seropositive patients. All specimens were processed with a commercially-available ELISA to detect C. parvum specific coproantigen and with a modified Ziehl-Neelsen stain (ZNm) microscope exam. Overall, sensitivity of the immunoenzymatic test was 100%, with a specificity of 96%; positive and negative predictive values were 89% and 100%, respectively. The commercial ELISA and ZNm proved to be valuable diagnostic tools for Cryptosporidium infection.

AIDS-Related Opportunistic Infections↗
PubMed1991

A simple method for cloning Giardia duodenalis from cultures and fecal samples.

Using a novel method for cloning Giardia duodenalis from cultures and fecal samples, 47 clones from 7 isolates were established in vitro. Average colony-forming efficiency in established cultures was 43.2% compared to 11.2% when cloning directly from excystation. The highest success rate of cloning was found with the Portland (P1, ATCC No. 30888) isolate, with a colony-forming efficiency of 92.7%. Cloned and parent populations were compared over a range of 13 enzymes using starch gel electrophoresis. No genetic difference was found between any of the clones and the parent isolates.

Animals↗
PubMed1996

Detection and molecular typing of Campylobacter jejuni in fecal samples by polymerase chain reaction.

In order to determine whether polymerase chain reaction (PCR) could be used to detect Campylobacter jejuni directly in stool samples, DNA from 66 frozen culture positive and negative fecal samples was purified by column chromatography. The flaA gene was amplified using primers directed against the conserved 5' and 3' regions and produced a 1.7 kb amplicon. Fifteen of 18 (83%) C. jejuni culture positive samples were detected by agarose gel electrophoresis and ethidium bromide staining. The test was negative in one sample containing C. coli. Twelve samples containing other enteric pathogens were negative as were 34 of 35 culture negative samples. Flagellin gene typing (see reference 14) of the flaA gene product from two stool samples in which the patients' stool isolate was also available showed the identical flagellin gene types suggesting that molecular typing of Campylobacter could potentially be performed on stool samples without the need for culture.

Bacterial Typing Techniques↗