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PubMed1999

Evaluation of quantitative latex agglutination for detection of Cryptosporidium parvum, E. coli K99, and rotavirus in calf feces.

A new methodology for detection of rotavirus, Escherichia coli K99, and Cryptosporidium parvum in bovine fecal samples was developed based on a quantitative latex agglutination technique (QLAT). Calibrated microspheres coated with specific antibodies to 1 of the enteric pathogens are quantitatively agglutinated by the antigens present in diluted fecal sample. The test is performed in a 96-well flat-bottom plate. The samples were tested with a commercial enzyme-linked immunosorbent assay kit prior to being analyzed by QLAT. The calculated sensitivity and specificity are adequate for field conditions, because the amount of the pathogenic agents is generally high. The overall time to perform the test was about 20 minutes.

Animals↗
PubMed1992

Evaluation of four serological tests for bovine paratuberculosis.

The standard complement fixation (CF) test, a commercial agarose gel immunodiffusion (AGID) test (ImmuCell Corporation, Portland, Maine), and two commercial enzyme-linked immunosorbent assays (ELISAs; Allied Laboratories, Glenwood Springs, Colo. [Allied ELISA], and the CSL, Limited, [Parkville, Victoria, Australia] enzyme immunoassay [CSL ELISA]) were evaluated by using sera from the Repository for Paratuberculosis Specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of dairy cattle without diarrhea or weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. None of the cattle evaluated had been vaccinated for paratuberculosis. Among 177 M. paratuberculosis-infected cattle, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA had test sensitivities of 38.4, 26.6, 58.8, and 43.4%, respectively, and specificities of 99.0, 100.0, 95.4, and 99.0%, respectively. Only 108 of the infected cattle were confirmed by culture or by a commercial DNA probe (IDEXX Corporation, Portland, Maine) to be shedding the organism in fecal samples at the time of serological testing. Among the 108 M. paratuberculosis fecal shedders, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA were positive for 54.6, 40.7, 65.7, and 56.5% of the cows, respectively; and among the 69 cows that were nonshedders, the tests were positive for 14.5, 4.3, 47.8, and 24.6% of the cows, respectively. There was a significant difference (P < 0.05) in the rate of positive test results for all four tests between these two groups of animals. The CF test performed well when it was compared with the other serological tests if a titer of >/- 1.8 was classified as a positive test result.

Animals↗
PubMed2003

Rapid gut transit time and slow fecal isoflavone disappearance phenotype are associated with greater genistein bioavailability in women.

The bioavailability of soybean isoflavones varies widely among individuals due to many factors, including activities of gut microflora. To characterize factors that affect fecal isoflavone disappearance phenotype and isoflavone bioavailability in women, 35 Asian and 33 Caucasian women, 18-43 y of age, provided fecal samples for anaerobic incubation with isoflavones in vitro at two times 5 mo apart (Phases I and II). Diet, physical activity and health history were investigated at these times. A single dose of soymilk powder [1.2 mg (4.57 micromol) total isoflavone/kg body] was given to all subjects with breakfast in phase II. Daidzein and genistein from fecal incubations, urine and fecal samples were measured by reverse-phase HPLC. Three significantly different daidzein and two genistein disappearance phenotypes were identified from fecal isoflavone incubations. More Asians than Caucasians were identified within the high daidzein disappearance phenotype. Caucasians and Asians differed significantly in daily intake of red meat (0.3 +/- 0.1 vs. 1.0 +/- 0.1 servings/d), dairy foods (2.9 +/- 0.2 vs. 1.2 +/- 0.2 servings/d) and insoluble dietary fiber (3.6 +/- 0.3 vs. 1.4 +/- 0.3 g). BMI, maximal oxygen uptake (VO(2 max)) and physical activity level were significantly greater in Caucasians than in Asians. Asian subjects of the low genistein disappearance phenotype had more rapid gut transit time (GTT) and greater isoflavone bioavailability as reflected in urinary genistein excretion than did Asians of the high genistein disappearance phenotype (GTT, 40 +/- 8 vs. 63 +/- 5 h; 11.0 +/- 2.7 vs. 4.0 +/- 1.7% of ingested genistein excreted in urine). Caucasians of both genistein disappearance phenotypes had longer GTT than did Asian subjects (84 +/- 5 vs. 56 +/- 6 h) and resembled Asians of the high genistein disappearance phenotype in genistein bioavailability. Relatively rapid GTT coupled with a low fecal isoflavone disappearance phenotype as occurred in Asian but not Caucasian subjects produced greater genistein bioavailability, as reflected in urinary genistein excretion.

Adolescent↗
PubMed1999

Seasonal enumeration of fecal coliform bacteria from the feces of ring-billed gulls (Larus delawarensis) and Canada geese (Branta canadensis).

Water suppliers have often implicated roosting birds for fecal contamination of their surface waters. Geese and gulls have been the primary targets of this blame although literature documenting the fecal coliform content of these birds is quite limited. To determine the actual fecal coliform concentrations of these birds, fecal samples from 249 ring-billed gulls and 236 Canada geese in Westchester County, N.Y., were analyzed over a 2-year period. Results indicate that gull feces contain a greater average concentration of fecal coliform bacteria per gram (3.68 x 10(8)) than do goose feces (1.53 x 10(4)); however, average fecal sample weights of the geese were more than 15 times higher than those of the gulls.

Animals↗
PubMed1987

[Epizootiology, prevention and control of salmonellosis in swine].

Microbiologic studies were carried out in 1981-1985 with a total of 25,561 samples--12,878 were cadavers and viscera, 574 were fetuses, 6,208 were swabs of rectal samples. 844 were feces, 4,096 were washings, 493--feed samples, etc. A number of prophylaxis and control programmes were tested under productional conditions with the use of drugs and vaccinations with a live and a killed autovaccine. Salmonella organisms were isolated as follows: 9.05% from cadavers, 2.5% from fecal samples, 1.35% from rectal swabs, 1.2% from washings from the environment, 0.6% from feed samples--all belonging to 19 serologic types. Greatest importance was shown to have S. cholerae suis, which was epizootically specific for swine, however, the part played by var. america also grew as compared to var. kunzendorf. With the exception of several sporadic cases all other serologic types of Salmonellae were isolated from carriers, rectal and fecal samples, and others. In the event of swine salmonellosis under productional conditions better results were obtained with the use of drugs. Both morbidity and mortality rate dropped twice as low with animals treated with a live vaccine once, and with a killed autovaccine four times. Suggested is a prophylactic and control program, following a definite pattern in dealing with swine salmonellosis in enzootic foci, with the use of complex measures.

Animals↗
PubMed2006

Clinical field efficacy and safety of pyrantel pamoate paste (19.13% w/w pyrantel base) against Anoplocephala spp. in naturally infected horses.

Clinical field trials were conducted at five geographical locations in the USA (Oklahoma, Wisconsin, Tennessee, Virginia and Idaho) to evaluate the efficacy and safety of pyrantel pamoate paste (19.13%, w/w, pyrantel base) administered at the recommended dosage of 13.2 mg pyrantel base/kg (6.0 mg pyrantel base/lb) body weight (b.w.) against tapeworm infections of Anoplocephala spp. in naturally infected horses. Horses at each study site were allocated by restricted randomization based on the cestode status (positive or negative) of pre-treatment fecal egg counts to complete sets of four animals each or incomplete sets of fewer than four animals. Within sets comprising of two to four horses, one animal was randomly allocated to receive placebo vehicle paste and the remaining horse(s) received pyrantel pamoate paste administered orally at a minimum dosage of 13.2 mg pyrantel base/kg b.w. on Test Day (TD) 0. Single animal sets received pyrantel pamoate paste. Fecal samples of horses were collected and examined for equine tapeworm (Anoplocephala spp.) eggs a minimum of four times (once or thrice between TD -28 and -14, twice between TD -14 and -7, and once on TD 0) prior to treatment on TD 0. Fecal samples of horses that were positive for cestode infection pre-treatment were examined for cestode eggs on TD 7, 8, 9, 14, 15 and 16. Cestode-negative pre-treatment horses were not sampled again after treatment. A total of 241 horses (141 mares, 16 stallions and 84 geldings; 6 months-30 yrs of age; 173-646 kg; 13 recognized breeds and various crossbreds) were evaluated. The prevalence of Anoplocephala spp. determined by pre-treatment fecal examination ranged from 38.3% in Idaho to 68.1% in Tennessee with an overall prevalence of 52.3%. Ninety cestode-positive and 88 cestode-negative horses were treated with pyrantel pamoate paste, 36 cestode-positive and 27 cestode-negative horses were treated with placebo vehicle paste. Overall, 178 horses were treated with pyrantel pamoate paste, and 63 horses were treated with placebo paste. Of the 178 horses treated with pyrantel pamoate paste, no drug related, adverse clinical or neurological health events were observed. No doses of pyrantel pamoate paste were refused or lost during dosing. At each post-treatment time sampling interval, significantly fewer cestode eggs (P < 0.0115) were passed by cestode-positive horses treated with pyrantel pamoate paste compared to cestode-positive horses that received placebo paste. Efficacy of the pyrantel pamoate paste treatment ranged from 92 to 96% from TD 7 to TD 16 with an overall efficacy of 95%. The results of these trials demonstrated that pyrantel pamoate paste (19.13%, w/w, pyrantel base) administered orally at a dosage of 13.2 mg pyrantel base/kg b.w. is highly efficacious (95%) against Anoplocephala spp. and safe for use in horses with no adverse clinical or neurological health events observed under field use conditions.

Administration, Oral↗
PubMed1995

Field application of polymerase chain reaction diagnosis and strain typing of Trypanosoma cruzi in Bolivian triatomines.

A new approach for direct identification and characterization of Trypanosoma cruzi stocks in biological samples was tested for field applicability on an extensive sample of feces collected from triatomine vectors from four different species found in Bolivia. The first step of the technique is polymerase chain reaction (PCR) amplification of the hypervariable region of kinetoplast DNA minicircles of T. cruzi parasites. In this report, 345 fecal samples were analyzed and the PCR results were compared with microscopic examination. For Triatoma infestans, the principal Bolivian vector, both techniques were in concordance 85.3% of the time. For the three other species, Rhodnius pictipes, Eratyrus mucronatus, and Triatoma sordida, the fecal samples were all negative by microscopic examination whereas PCR results showed several T. cruzi-infected insects in each species. The second step of the procedure is the characterization of the T. cruzi clones by means of hybridization of the PCR products with clone-specific probes generated by the PCR. We used two probes corresponding to major clones circulating in high frequency in Bolivia (as shown by previous population genetic studies using isoenzyme characterization). We obtained four primary results: 1) we confirm the importance of two major clones in Bolivia in two distinct regions; 2) we report high rates of mixed infections (multiple clones in a single vector) in Triatoma infestans, up to 22% and 35% in Cochabamba and La Paz departments, respectively; 3) the results favor the absence of interaction between different clones; and 4) we find, for the first time, evidence of the major clones circulating in three species of triatomines that are known as mainly sylvatic species.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Gastrointestinal carriage of toxigenic bacteria: relation to diarrhea and to serum immune response.

We followed up a cohort of babies from birth to two years by examination of fecal samples for heat-labile enterotoxigenic (LT+) bacteria and administration of a questionnaire about any episodes of diarrhea each week. We sampled serum at birth, every six months, at the beginning of an episode of diarrhea, and two weeks later. We tested sera for antibody to cholera toxin. We identified LT+ bacteria in 16% of fecal samples, LT+ Escherichia coli in 11%. Thirty-four episodes of diarrhea occurred within a week of isolation of LT+ bacteria. Bacterial species and weeks of exposure to LT+ bacteria correlated with diarrhea. Fourfold rises in antibody titer followed 53% of diarrhea episodes caused by LT+ bacteria. Whether a baby had an immune response was not related to his age, duration of diarrhea, cord serum antibody, previous asymptomatic carriage of LT+ bacteria, or breast-feeding. Second episodes did not boost antibody levels. Rises in antibody titers followed diarrhea without the presence of toxin but did not follow asymptomatic carriage of LT+ bacteria.

Age Factors↗
PubMed2003

Evaluation of the 1-2 test for detecting Salmonella in swine feces.

The 1-2 Test is a rapid culture test for the detection of motile Salmonella. The aim of this study was to evaluate the 1-2 Test for its ability to detect Salmonella in swine feces following preenrichment and selective enrichment. Pooled pen fecal samples (n = 118) and pig rectal swabs (n = 51) were cultured for Salmonella by the 1-2 Test, which was compared with the standard isolation protocol currently used in our laboratory. In addition, pen fecal samples known to be free of Salmonella were spiked with various concentrations of Salmonella enterica subsp. enterica serovar Typhimurium and cultured by both methods to determine the minimum number of organisms needed to produce a positive result. When naturally contaminated pen feces and rectal swabs were used, results obtained with the standard culture method were similar to those obtained with the 1-2 Test. However, the 1-2 Test did outperform the standard culture method when the spiked feces samples were tested (chi2 = 4.00). The test kit reduced the time and materials required for the detection of Salmonella in swine feces. The results of this study indicate that the 1-2 Test is an accurate method for monitoring Salmonella in swine feces.

Animals↗
PubMed1985

[Rotavirus and pararotavirus infections in adults. Analysis of a nosocomial infection and some sporadic cases].

A nosocomial infection in adults due to rotavirus is described. The identity of the infecting rotavirus strains was demonstrated by genomic dsRNA electrophoresis. At an interval of 19 days another patient in the same hospital developed a pararotavirus infection. Pararotaviruses or rotavirus-like agents are antigenetically distinct from rotaviruses and thus cannot be detected at present by the available antibody mediated antigen detection systems. The current prevalence of pararotavirus infections in adults is unknown in Switzerland. The question whether there is a relationship between the small nosocomial rotavirus epidemic and the sporadic pararotavirus infection remains speculative. We also performed microbiological workups of 394 fecal samples from outpatients with diarrhoea. During the study period from January to April 1984 rotaviruses were the third most frequent aetiological agent, after salmonella and campylobacter, in acute adult gastroenteritis. Selecting the bacteriologically negative fecal samples with a watery and non-bloody aspect, rotaviruses were found in 30%.

Adult↗
PubMed1994

Sensitive detection of canine parvovirus DNA by the nested polymerase chain reaction.

A polymerase chain reaction (PCR) for the detection of canine parvovirus (CPV) was developed. To increase the sensitivity and specificity of the reaction, the nested PCR with a double-nested primer pair (inner primer pair) was designed. The sequences of the PCR primer pairs were selected from the conserved region in the CPV VP1/VP2 gene. The PCR with the outer or inner primer pair alone (single PCR) could detect 10 fg of viral replicative form (RF) DNA on agarose gel electrophoresis; whereas as little as 100 ag of the RF DNA was detected by the nested PCR, which was shown to be 100 times more sensitive than the single PCR. Samples prepared from feline panleukopenia virus and mink enteritis virus, both having a very close antigenic relationship to CPV, were also amplified by the nested PCR. The specificity of the reaction was confirmed by restriction enzyme analysis and Southern hybridization. Next, fecal samples were examined by the nested PCR. All 10 samples suspected of CPV infection were positive, and two restriction sites (HaeIII and HindIII sites) in the PCR product were conserved among them. On the other hand, specific amplification was not observed in the samples derived from normal dogs. The number of the genome copy in positive samples was estimated about 10(9)-10(11)/g by the single PCR and 10(11)-10(13)/g by the nested PCR. The assay can be completed in 1-1.5 days, and does not need radioisotopes. Thus, the nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in fecal samples.

Animals↗
PubMed2004

Outcome of cats with diarrhea and Tritrichomonas foetus infection.

OBJECTIVE: To determine the long-term outcome of cats infected with Tritrichomonas foetus and identify treatment and management strategies influencing resolution of infection or associated diarrhea. DESIGN: Prospective study. SAMPLE POPULATION: 26 cats with T. foetus-associated diarrhea at least 22 months prior to the study. PROCEDURE: A standardized survey regarding clinical course and management was administered to owners of cats with T. foetus infection and associated diarrhea. Fecal samples were obtained from each cat; the presence of T. foetus was assessed via microscopic examination of smears, culture in commercial media, and polymerase chain reaction amplification of T. foetus rDNA involving species-specific primers. RESULTS: Survey responses were obtained from owners of all 26 cats. Twenty-three cats had complete resolution of diarrhea a median of 9 months after onset. Analysis of fecal samples obtained from 22 cats revealed persistent T. foetus infection in 12, with a median of 39 months after resolution of diarrhea. History of implementation of a dietary change, treatment with paromomycin, or higher numbers of cats in the household was associated with significantly longer duration of time to resolution of diarrhea. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested chronic T. foetus-associated diarrhea in most cats is likely to resolve spontaneously within 2 years of onset. Chronic infection with T. foetus (without clinical signs) after resolution of diarrhea appears to be common. Although often temporarily effective in decreasing severity of diarrhea, attempts to treat cats with T. foetus infection may result in prolongation of time to resolution of diarrhea.

Animals↗
PubMed2005

Experience from the development of a diagnostic single tube real-time PCR for human caliciviruses, Norovirus genogroups I and II.

Detection of caliciviruses requires high mutation tolerance and throughput. The development of a rational simple, single tube reverse transcription-real-time quantitative PCR (QPCR) technique for human noroviruses (NV) is reported here. A dual-probe, triple-primer system (NM system) was used for simultaneous detection and preliminary differentiation of NV genogroups in fecal samples. The design was based on a comprehensive analysis of all 1140 NV sequences available in GenBank. A touch-down amplification protocol improved the frequency of detection. The final QPCR was evaluated with 71 fecal samples from outbreak and sporadic cases in Sweden (1997-2004), all calicivirus-positive by electron microscopy. Up to 56 (79 %) were positive. The method is more rational than NV detection methods described previously, and should be a developmental basis for large-scale routine methods for detection of NV.

Adult↗
PubMed2006

Virulence profiles of Shiga toxin 2e-producing Escherichia coli isolated from healthy pig at slaughter.

Recently, virulence patterns of Stx2e-producing Escherichia coli from pigs with edema disease and from humans were compared and strains from diseased pigs were reported to be unlikely human pathogens [Sonntag, A.K., Bielaszewska, M., Mellmann, A., Dierksen, N., Schierack, P., Wieler, L.H., Schmidt, M.A., Karch, H., 2005. Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8855-8863]. In the present study, 31 Shiga toxin-producing E. coli (STEC) strains harboring stx2e, which were previously isolated out of fecal samples from healthy pigs at slaughter [Kaufmann, M., Zweifel, C., Blanco, M., Blanco, J.E., Blanco, J., Beutin, L., Stephan, R., 2006. Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland. J. Food Prot. 69, 260-266], were characterized by phenotypic and genotypic traits. Nine of the thirty-one sorbitol-positive non-O157 STEC (stx2e) isolated from healthy pigs belonged to serotypes found in STEC isolated from humans, including two serotypes (O9:H-, O26:H-) reported in association with hemolytic-uremic syndrome. Otherwise, the serotypes were different from those isolated from cases of edema disease in pigs. The eae (intimin) gene, which is strongly correlated with severe human disease, was not detected. Moreover, all strains were lacking the genes for enterohemolysin (ehxA), porcine A/E associated protein (paa), STEC autoagglutinating adhesin (saa) and the serin protease EspI (espI). Nine strains tested positive for astA (EAST1), one O141:H17 strain for fedA (F18 fimbrial adhesin) and one O159:H- strain for terF (tellurite resistance). Similar to the Stx2e-producing E. coli isolated from humans, which are mainly lacking further virulence factors, genes of an iron uptake system on the high-pathogenicity island (irp2, fyuA) were detected in three ONT:H10 and ONT:H19 strains from healthy pigs. Consequently, although the isolated strains are unlikely to be associated with severe human diseases, healthy pigs cannot be excluded as a potential source of human infection with Stx2e-producing STEC.

Abattoirs↗
PubMed2002

Gastrointestinal tract location of Escherichia coli O157:H7 in ruminants.

Experimentally inoculated sheep and cattle were used as models of natural ruminant infection to investigate the pattern of Escherichia coli O157:H7 shedding and gastrointestinal tract (GIT) location. Eighteen forage-fed cattle were orally inoculated with E. coli O157:H7, and fecal samples were cultured for the bacteria. Three distinct patterns of shedding were observed: 1 month, 1 week, and 2 months or more. Similar patterns were confirmed among 29 forage-fed sheep and four cannulated steers. To identify the GIT location of E. coli O157:H7, sheep were sacrificed at weekly intervals postinoculation and tissue and digesta cultures were taken from the rumen, abomasum, duodenum, lower ileum, cecum, ascending colon, descending colon, and rectum. E. coli O157:H7 was most prevalent in the lower GIT digesta, specifically the cecum, colon, and feces. The bacteria were only inconsistently cultured from tissue samples and only during the first week postinoculation. These results were supported in studies of four Angus steers with cannulae inserted into both the rumen and duodenum. After the steers were inoculated, ruminal, duodenal, and fecal samples were cultured periodically over the course of the infection. The predominant location of E. coli O157:H7 persistence was the lower GIT. E. coli O157:H7 was rarely cultured from the rumen or duodenum after the first week postinoculation, and this did not predict if animals went on to shed the bacteria for 1 week or 1 month. These findings suggest the colon as the site for E. coli O157:H7 persistence and proliferation in mature ruminant animals.

Animals↗
PubMed1995

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique. The other strain commonly encountered among the neonates was also identified frequently among the isolates from patients with antibiotic-associated diarrhea or pseudomembranous colitis, indicating its general occurrence. On the basis of both epidemiological studies and PCR-mediated genotyping, it was shown that the environment and not the birth canal is the major source of C. difficile acquisition by neonates in this maternity hospital setting. Furthermore, AP-PCR appears to be a fast and useful method for epidemiologically relevant typing of C. difficile isolates.

Bacterial Typing Techniques↗
PubMed2006

Detection of Mycobacterium avium subsp. paratuberculosis in bovine manure using Whatman FTA card technology and Lightcycler real-time PCR.

A modified forensic DNA extraction and real-time fluorescent polymerase chain reaction assay has been evaluated for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine fecal samples using primers and fluorescent resonance energy transfer (FRET) probes targeting the IS900 gene sequence of MAP. DNA was successfully extracted from manure samples by utilizing the Whatman FTA card technology, which allows for simple processing and storage of samples at room temperature. The FTA cards were washed and subjected to a Chelex-100 incubation to remove any remaining polymerase chain reaction (PCR) inhibitors and to elute the DNA from the FTA card. This isolated DNA was then subjected to direct real time fluorescent PCR analysis. Detection of MAP DNA from bovine fecal samples spiked with known concentrations of viable MAP cells was obtained. The detection limits of the assay was consistently found to be between 10(2) and 10(4) colony forming units [CFU]/g, with some samples containing as low as 10 CFU/g, yielding positive assay results. This cost-efficient assay allows reporting of results as early as 4 h after fecal collection, which can be particularly useful in highthroughput herd screening.

Animals↗
PubMed2005

Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula.

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5' nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5' nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% +/- 9.8% to 73.4% +/- 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% +/- 1.92% and 8.11% +/- 4.12%, respectively, versus 0.15% +/- 0.11% and 1.38% +/- 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.

Bifidobacterium↗