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Evaluation of a PCR to detect Salmonella in fecal samples of horses admitted to a veterinary teaching hospital.

The diagnostic accuracy of a PCR used to identify horses shedding Salmonella spp. in their feces during hospitalization was estimated, relative to bacterial culture of serially collected fecal samples, using longitudinal data. Five or more fecal samples were collected from each of 116 horses admitted as inpatients, for reasons other than gastrointestinal disease, between July 26, 2001 and October 25, 2002. All 873 fecal samples collected were tested with a PCR based on oligonucleotide primers defining a highly conserved segment of the histidine transport operon gene of Salmonella typhimurium, and each sample was cultured for Salmonella spp. One or more samples from 87 (75%) horses were PCR positive, and Salmonella was cultured from 1 or more samples from 11 (9.5%) horses. All culture-positive horses had at least 1 PCR-positive result, whereas only 29 (28%) culture-negative horses were PCR negative on all fecal samples tested. The PCR was most specific, relative to bacterial culture of serially collected fecal samples, when used to test samples from Quarterhorse or breeds other than Thoroughbred or Standardbred, or from clinical (vs. healthy, accompanying horses) cases. Overall, the PCR had the greatest agreement (70%), compared with bacterial culture of serially collected fecal samples, using a cutoff of 2 or more positive PCR test results to define a Salmonella-positive horse. The reasons why some fecal samples, from which Salmonella organisms cannot be isolated, are PCR positive need to be determined before the PCR can be incorporated into Salmonella surveillance programs for hospitalized equine populations.

Animals↗

Comparison of results using electron microscope, immunodiffusion and fluorescent antibody analyses to detect rotavirus in diarrheic fecal samples of calves.

Seventy-nine diarrheic calf fecal samples were examined by electron microscopy, immunodiffusion and the fluorescent antibody technique for the presence of rotavirus (reovirus-like agent). Thirty-eight (48%) of the samples were positive by electron microscopy, 59% by immunodiffusion and 20% positive by fluorescent antibody technique analyses. Another 9% were suspect-positive by fluorescent antibody technique. Chymotrypsin treatment of the fecal samples increased the ease of observing the viral particles by electron microscopy and also intensified the immunodiffusion arcs obtained. Immunodiffusion analyses using specific antisera to the virus would appear to be a practical method of detecting rotavirus in diarrheic fecal samples.

Animals↗

[The occurrence of Prototheca in fecal samples of cattle].

Prototheca spp. were detected in 146 (48.7%) of culturally examined fecal samples of cattle, using the selective medium, developed by Pore (1973). 114 fecal samples (78.1%) contained a monoculture of Prototheca (P.) zopfii, while 10 fecal samples (6.8%) contained a monoculture of P. moriformis. In further 22 fecal samples (15.1%) a mixed culture of P. zopfii and P. moriformis was detected. The results of this study permit to conclude that cattle can harbour and shed Prototheca spp. in variable frequency.

Animals↗

Detection of Cryptosporidium oocysts in canine fecal samples by immunofluorescence assay.

Fecal samples were collected from 257 dogs in four areas in Korea during the period of January 1996 to November 1997 and examined by immunofluorescence assay for Cryptosporidium oocysts using a commercial diagnostic kit (Meridian Diagnostics, Cincinnati, Ohio). Of the 257 samples, 25 (9.7%) were positive for Cryptosporidium. Differences were noted in the prevalence of canine cryptosporidiosis in both areas and dog types. The results provide a further evidence of environmental contamination and widespread distribution of the parasite in Korea.

Animals↗

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples.

OBJECTIVE: To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. MATERIALS AND METHODS: Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-specific oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to confirm oxc. RESULTS: A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the amplified product was compatible with that of oxc. CONCLUSION: O. formigenes can be identified easily and efficiently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.

Adult↗

Evaluation of DNA probes for detection of Shiga-like-toxin-producing Escherichia coli in food and calf fecal samples.

The use of DNA probes for Shiga-like toxin I (SLT-I) and SLT-II for detection of SLT-producing Escherichia coli (SLTEC) in foods and calf fecal samples was evaluated. Enrichment cultures were prepared from food or fecal samples. Colonies formed by plating of enrichment cultures were probed for SLTEC by colony hybridization. Alternatively, enrichment cultures were analyzed for SLTEC presence by dot blot. The lowest detected concentration of SLTEC in sample homogenates inoculated with E. coli O157:H7 corresponded to 1.3 CFU/g of sample. Of the 44 food samples and 28 fecal samples from dairy calves tested by the colony hybridization method, 4 food samples, including ground beef, raw goat milk, blueberries, and surimi-based delicatessen salad, and 9 calf fecal samples were positive with the SLT probes. The dot blot technique yielded results within 48 h and can be used as a fast and sensitive method of detection for SLTEC in foods and calf fecal samples. The colony hybridization technique took 3 to 4 days but permits recovery of the positive colonies when desired.

Animals↗

Digoxigenin-labeled deoxyribonucleic acid probes for the enumeration of bifidobacteria in fecal samples.

The numbers of bifidobacteria in fecal samples were specifically determined by colony hybridization with the mixture of digoxigenin-labeled DNA probes that were prepared from whole chromosomal DNA of Bifidobacterium longum 6001 and Bifidobacterium adolescentis 6003. These DNA probes strongly hybridized with DNA of B. longum, B. adolescentis, Bifidobacterium breve, Bifidobacterium suis, Bifidobacterium infantis, Bifidobacterium bifidum, Bifidobacterium angulatum, and Bifidobacterium animalis. Detectable positive signals with DNA of Bifidobacterium pseudolongum ssp. pseudolongum, Bifidobacterium catenulatum, and Bifidobacterium thermophilum were also found after hybridization. When dot-blot hybridization was performed with whole cells of 47 reference strains containing 11 species (16 strains) of bifidobacteria, all of the bifidobacteria tested could be specifically detected by using these DNA probes; Lactobacillus fermentum JCM 1173, however, showed a slight nonspecific signal. The counts of bifidobacteria by colony hybridization in the fecal samples of four of the five subjects were the same as the counts that were obtained by the conventional method using BL agar medium. Furthermore, no significant difference existed in the number of bifidobacteria that were determined by either method.

Adult↗

An improved method for cultivation of Mycobacterium paratuberculosis from bovine fecal samples and comparison to three other methods.

A new method (NADC) for isolation of Mycobacterium paratuberculosis from fecal samples is described and evaluated using fecal samples from a known paratuberculosis-infected herd of cattle. The NADC method includes centrifugation of the total fecal sample supernatant and use of a 2-step decontamination protocol. The growth rate of M. paratuberculosis and contamination rate of cultures when using this method are compared to 3 other published methods: sedimentation, centrifugation, and Cornell. Sensitivity was lowest for the Cornell method precluding detection of some low shedders; however, contamination was not observed for this method. Contamination was the most severe in samples processed by the centrifugation method but was also high for the sedimentation method, resulting in unreadable culture tubes for some fecal samples. The NADC method was 10-fold more sensitive for detection of M. paratuberculosis colonies and contamination was significantly reduced compared to other 3 methods.

Animals↗

Detection of group B rotaviruses in fecal samples from diarrheic calves and adult cows and characterization of their VP7 genes.

Groups A, B, and C rotaviruses have been identified in cattle. Group B rotaviruses are associated with sporadic cases of diarrhea in calves and adult cows. From diagnostic submissions to our laboratory, 90 fecal samples from cases of calf diarrhea, 81 fecal samples from cases of adult cow diarrhea (winter dysentery), and 20 fecal samples from case control normal adult cows were tested for group B rotaviruses by polyacrylamide gel electrophoresis (PAGE), and reverse transcription (RT)-PCR (targeting 279 bp of the VP7 gene). In addition, 53 fecal samples from diarrheic adult cows were tested for group B rotaviruses by immune electron microscopy (IEM). By RT-PCR, five samples from calves were group B rotavirus positive (5.6%). Fifteen samples from adult cows with diarrhea were group B rotavirus positive (18.5%), and none of the control fecal samples from normal cows were positive for group B rotaviruses. By PAGE, one calf sample (RT-PCR positive) was group B rotavirus positive (short electropherotype), but none of the adult cow samples were positive for group B rotaviruses. By IEM, 5 (9.4%) of the 53 fecal samples from diarrheic adult cows were group B positive (all were also RT-PCR positive). The VP7 genes of three strains (WD653 from an adult cow and the ATI and Mebus calf strains) were sequenced. The VP7 genes from the three bovine strains showed high (over 90%) nucleotide and deduced amino acid homologies, but lower homologies (48 to 61%) were seen between these genes and the genes from rodent (IDIR) and human (ADRV) group B rotaviruses. Although there were some differences of degree, all inoculated gnotobiotic calves (n = 6) showed abnormal feces between 1 and 3 days after inoculation with each of three strains of group B bovine rotaviruses, and group B rotaviruse, were detected in the feces for up to 2 weeks by RT-PCR but for shorter periods by PAGE or IEM.

Animals↗

Effect of temperature on the detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus in fecal samples by reverse transcription-polymerase chain reaction.

The effect of storage temperature was determined for the detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in fecal samples from experimentally and naturally infected pigs by multiplex reverse transcription-polymerase chain reaction (RT-PCR). To examine the effect of storage temperature on the ability to detect PEDV and TGEV RNA by multiplex RT-PCR, fecal samples were stored for different temperatures (4, 21, 36, and 45 C) before extracting viral RNA. The PEDV and TGEV nucleic acids in fecal samples were stable up to 3 days at 4 C. The PEDV and TGEV nucleic acids were consistently detected in fecal samples up to 60 hours at 21 C and 24 hours only at 36 and 45 C. Thereafter, the number of positive signals declined. Positive signals were detected in fecal samples stored at 4 C by 240 hours. The PEDV and TGEV nucleic acids were consistently detected in fecal samples up to 60 hours at 21 C and 24 hours only at 36 and 45 C. The results of this study suggested that storage temperature has significant effect on the detection of PEDV and TGEV nucleic acids from fecal samples by multiplex RT-PCR.

Animals↗

Rapid detection of toxigenic Clostridium difficile in fecal samples by magnetic immuno PCR assay.

Rapid detection of toxigenic Clostridium difficile in fecal samples was accomplished with the magnetic immuno PCR assay (MIPA). Elaborate DNA extraction techniques were unnecessary. First, we generated a mouse monoclonal antibody (MAb) reactive with only C. difficile, Clostridium sordellii, and Clostridium bifermentans. Then, magnetic beads were coated with the MAb, incubated with fecal samples to allow binding with C. difficile, extracted from the stool with a magnet, and processed in the PCR with primers specific for the toxin B gene. After optimizing MIPA by raising the number of PCR cycles from 35 to 40 and adding Chelex 100 to the PCR mixture, we found a sensitivity of 96.7%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94.1% when compared with the culture of cytotoxic C. difficile from fecal samples. MIPA is a rapid, easy, and sensitive PCR method for demonstrating the presence of toxigenic C. difficile in stool samples and avoids the disadvantage of elaborate extraction of DNA from fecal samples.

Antibodies, Monoclonal↗

Occurrence of Cryptosporidium oocysts in fecal samples submitted for routine microbiological examination.

During a 7-month period, 2,252 fecal samples submitted for routine microbiological examination from 1,621 patients were screened for Cryptosporidium oocysts by the auramine staining method with Kinyoun acid-fast stain as the confirmatory stain. Cryptosporidium oocysts were detected in fecal samples from 19 (1.2%) patients, 18 of whom had gastroenteritis. Diarrheic stools from 14 of these 18 patients were negative for the usual enteropathogens but contained the oocysts in moderate to large numbers. Although Cryptosporidium oocysts were found in patients of all ages, they occurred slightly more frequently in infants and children than in the rest. Cryptosporidium species was one of the common enteropathogens identified in fecal samples submitted for routine parasitological examination during the period of the survey and was second only to Giardia species in terms of frequency. Considering cryptosporidiosis in the differential diagnosis of gastroenteritis in immunocompetent persons and including a search for Cryptosporidium oocysts in routine parasitological examinations of fecal samples appear warranted.

Adolescent↗

[Two genotypes of Canine coronavirus simultaneously detected in the fecal samples of healthy foxes and raccoon dogs].

61 fecal samples from healthy foxes and 24 from healthy raccoon dogs were examined by RT-nested PCR assays for the presence and genotypic identification of Canine coronaviruses (CCVs). 77.0% fox samples were recognized as CCV positive, 43 of which belonged to type II and 29 to type I, as well as both genotypes were simultaneously detected in 25 samples. Out of the total 24 fecal samples from raccoon dogs, 22 were CCV positive for type II and 16 for type I. M gene fragments of 8 samples were sequenced, 4 of which were confirmed as CCV type I and the other 4 as CCV type II. Sequence analysis showed that the M gene of CCV type I had a high similarity of 96.7% - 98.1% between the fox-and raccoon dog strains and the reported Italian strain from diarrhea dogs. The two genotypes, with an identity of 88.3% - 89.7%, formed two separate branches in phylogenetic tree. Interestingly, the sequence at several nucleic acid sites of CCV type II differed between foxes and raccoon dogs. The co-existence and popularity of the two CCV genotypes in healthy foxes and raccoon dogs were first confirmed in this article.

Animals↗

Development of a combined selection and enrichment PCR procedure for Clostridium botulinum Types B, E, and F and its use to determine prevalence in fecal samples from slaughtered pigs.

A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymerase rTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 x 10(3) spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

Abattoirs↗

A matter of time: evaluating the storage of fecal samples for steroid analysis.

The extraction and immunoassay of fecal steroids is an increasingly common technique, used in both captive and field studies to provide an approximation of an animal's circulating concentration of hormones through non-invasive methods. Storage of fecal samples is of critical concern because fecal bacteria metabolize fecal steroids within hours after deposit. Ethanol is often used as a preservative for fecal samples stored for several hours at room temperature. We examined the stability of fecal estrogen (fE) and glucocorticoid (fGC) metabolites from baboon (Papio cynocephalus) samples in a 95% ethanol solution at ambient temperature and at -20 degrees C over the course of six months, to determine the effect of storage on steroid concentrations. As measured by radioimmunoassay, fE metabolite concentrations increased by 122% at 90 days and fGC metabolite concentrations increased by 92% at 120 days. After peaking, both hormones declined to near initial concentrations by 180 days in ambient temperature samples. In samples stored at sub-zero temperatures, fGC metabolite concentrations showed a similar but dampened pattern, while fE metabolite concentrations exhibited small and variable changes with no consistent trend. We discuss explanations for the dynamic pattern of changing fecal metabolite concentrations and offer practical and analytical guidance to field workers for situations in which ideal conditions for stabilizing hormones are not available.

Animals↗

Prevalence and molecular characterization of Escherichia coli O157:H7 in bulk tank milk and fecal samples from cull cows: a 12-month survey of dairy farms in east Tennessee.

A study on the prevalence of Escherichia coli O157:H7 was conducted on 30 dairy farms in east Tennessee between May 2000 and April 2001. This pathogen was isolated from 8 of 30 (26.7%) dairy farms at various sampling times. A total of 415 fecal samples from cull dairy cows and 268 bulk tank milk samples were analyzed. Overall, 10 of 683 (1.46%) samples (2 of 268 [0.75%] milk samples and 8 of 415 [1.93%] fecal samples) tested positive for E. coli O157:H7. Food and Drug Administration Bacteriological Analytical Manual protocols were used for the conventional isolation and confirmation of E. coli O157:H7. Samples were shake cultured (150 rpm) at 42 degrees C for 24 h in tryptic soy broth containing 2 mg of novobiocin per liter. White colonies isolated on cefixime-tellurite sorbitol MacConkey agar plates were evaluated for fluorescence on sorbitol MacConkey agar supplemented with 0.025 g of methylumbelliferyl-beta-D-glucuronide per liter. Nonfluorescing white colonies were biochemically typed and serologically confirmed. Multiplex polymerase chain reaction profiles of E. coli O157:H7 isolates indicated the presence of common virulence factors (Shiga toxin, enterohemolysin, and intimin) of Shiga toxin-producing E. coli, suggesting the potential human pathogenicity of bacterial isolates. Pulsed-field gel electrophoresis profiles of SpeI and XbaI restriction enzyme-digested genomic DNA were used to establish relatedness among bacterial isolates. Data from this study indicate that both cull dairy cows and bulk tank milk pose a potential hazard with regard to human foodborne illness. It is therefore imperative to develop on-farm and preharvest pathogen reduction programs to control the carriage of E. coli O157:H7 pathogens.

Animals↗

Comparison of fecal samples collected per rectum and off the ground for estimation of environmental contamination attributable to beef cattle.

OBJECTIVES: To determine whether sampling feces off the ground replicates prevalence estimates for specific pathogens obtained from fecal samples collected per rectum of adult cows, and to determine characteristics of feces on the ground (fecal pats) that are associated with subsequent identification of Campylobacter spp, Cryptosporidium parvum, and Giardia duodenalis. ANIMALS: A random sample of adult beef cattle from 25 herds located throughout California. PROCEDURE: 1,115 rectal and ground fecal samples were obtained. Samples were submitted for culture of Campylobacter spp and examined, using a direct fluorescent antibody assay, to detect C parvum oocysts and G duodenalis cysts. Characteristics of fecal pats, such as volume and consistency, were recorded. RESULTS: Prevalence of Campylobacter spp was 5.0% (20/401) for rectal fecal samples, which was significantly greater than prevalence determined for ground fecal samples (2/402; 0.5%). Most isolates were C jejuni subsp jejuni. Prevalence of C parvum was higher in rectal fecal samples (6/557; 1.1%) than in ground fecal samples (1/558; 0.2%), but this difference was not significant. Prevalence of G duodenalis did not differ for rectal (36/557; 6.5%) versus ground (26/558; 4.7%) fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of ground fecal samples may not accurately indicate the prevalence of Campylobacter spp or C parvum in cattle but may reflect prevalence of G duodenalis. Differences in prevalence estimates between the 2 methods suggest inactivation of pathogens in feces after cattle have defecated. Prevalence estimates generated by evaluation of ground fecal samples, however, may more accurately estimate environmental pathogen burden.

Animals↗

Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota: detection of intestinal trophozoites at necropsy provides greater sensitivity than detection of cysts in fecal samples.

Surveys of the prevalence of the intestinal protozoan Giardia spp. in animal populations have relied almost exclusively on the detection of cysts in fecal samples. We have determined the prevalence of Giardia spp. in beaver and muskrat populations in four northeastern states and Minnesota by using both the detection of trophozoites in mucosal scrapings from live-trapped animals at necropsy and the detection of cysts in fecal samples collected from kill-trapped animals. In muskrats the prevalence of Giardia infection was 36.6% by cyst detection in fecal samples (n = 790) from kill-trapped animals and 95.9% in live-trapped muskrats when the intestinal contents were analyzed for the presence of trophozoites (n = 219). Similarly, in beavers, Giardia infection was 9.2% by cyst detection in fecal samples (n = 662) from kill-trapped beavers and 13.7% in live-trapped animals examined for the presence of intestinal trophozoites (n = 302). The detection of trophozoites in mucosal scrapings from live-trapped animals consistently yielded a significantly higher prevalence for both muskrats and beavers than did the method based on detection of cysts in the fecal samples. The prevalence of Giardia infection in juvenile and adult live-trapped muskrats was similar (92.5 and 94.4%, respectively), but the prevalence in juvenile live-trapped beavers (23.2%) was significantly greater than that seen in the adult animals (12.6%). No difference in Giardia prevalence on the basis of sex was seen in either animal species. Regional variation, often statistically significant, was seen in the prevalence of Giardia in beavers in the northeastern states and Minnesota, but was not detected for muskrats.

Animals↗