Identification of Bifidobacterium species using rep-PCR fingerprinting

The aim of the present study was to evaluate the use of repetitive DNA element PCR fingerprinting (rep-PCR) for the taxonomic discrimination among the currently described species within the genus Bifidobacterium. After evaluating several primer sets targeting the repetitive DNA elements BOX, ERIC, (GTG)s and REP, the BOXA1R primer was found to be the most optimal choice for the establishment of a taxonomical framework of 80 Bifidobacterium type and reference strains. Subsequently, the BOX-PCR protocol was tested for the identification of 48 unknown bifidobacterial isolates originating from human faecal samples and probiotic products. In conclusion, rep-PCR fingerprinting using the BOXA1R primer can be considered as a promising genotypic tool for the identification of a wide range of bifidobacteria at the species, subspecies and potentially up to the strain level.     

An evaluation of long-term preservation methods for brown bear (Ursus arctos) faecal DNA samples

Relatively few large-scale faecal DNA studieshave been initiated due to difficulties inamplifying low quality and quantity DNAtemplate. To improve brown bear faecal DNA PCRamplification success rates and to determinepost collection sample longevity, fivepreservation methods were evaluated: 90%ethanol, DETs buffer, silica-dried, oven-driedstored at room temperature, and oven-driedstored at –20 °C. Preservationeffectiveness was evaluated for 50 faecalsamples by PCR amplification of a mitochondrialDNA (mtDNA) locus (146 bp) and a nuclear DNA(nDNA) locus (200 bp) at time points of oneweek, one month, three months and six months. Preservation method and storage timesignificantly impacted mtDNA and nDNAamplification success rates. For mtDNA, allpreservation methods had 75% success atone week, but storage time had a significantimpact on the effectiveness of the silicapreservation method. Ethanol preserved sampleshad the highest success rates for both mtDNA(86.5%) and nDNA (84%). Nuclear DNAamplification success rates ranged from 26–88%, and storage time had a significant impacton all methods but ethanol. Preservationmethod and storage time should be importantconsiderations for researchers planningprojects utilizing faecal DNA. We recommendpreservation of faecal samples in 90% ethanolwhen feasible, although when collecting inremote field conditions or for both DNA andhormone assays a dry collection method may beadvantageous.     

Characterization of faecal enterococci from rabbits for the selection of probiotic strains

AIMS: To characterize the facultative anaerobic intestinal microbiota of healthy rabbits, especially enterococci, for the selection of potential probiotic strains. METHODS AND RESULTS: Phenotypic and molecular methods were used to identify enterococcal isolates. Results obtained indicated that enterococcal microbiota widely varied among individuals both in size and in composition. Antibacterial and haemolytic activities, and resistance to acid and bile salts were determined. A small group of strains produced bacteriocins active against listeriae and indigenous clostridia and therefore they were selected as potential probiotics. One such strain, 8G, was assayed for colonization capacity. Results obtained suggested that the fate of the introduced strain depended on the composition of the enterococcal indigenous microbiota. CONCLUSIONS: Enterococcus faecalis and Ent. faecium are the predominant enterococcal species in the gut of rabbits. Other species of lactic acid bacteria were not recovered. SIGNIFICANCE AND IMPACT OF THE STUDY: The enterococcal intestinal microbiota of healthy rabbits has been characterized in detail. Monitoring the fate of an introduced probiotic in vivo is required in order to evaluate potential probiotic strains.     

Molecular tracking of mountain lions in the Yosemite valley region in California: genetic analysis using microsatellites and faecal DNA

Twelve microsatellite loci were characterized in California mountain lions (Puma concolor) and sufficient polymorphism was found to uniquely genotype 62 animals sampled at necropsy. Microsatellite genotypes obtained using mountain lion faecal DNA matched those from muscle for all of 15 individuals examined. DNA from potential prey species and animals whose faeces could be misidentified as mountain lion faeces were reliably distinguished from mountain lions using this microsatellite panel. In a field application of this technique, 32 faecal samples were collected from hiking trails in the Yosemite Valley region where seven mountain lions previously had been captured, sampled, and released. Twelve samples yielded characteristic mountain lion genotypes, three displayed bobcat-type genotypes, and 17 did not amplify. The genotype of one of the 12 mountain lion faecal samples was identical to one of the mountain lions that previously had been captured. Three of the 12 faecal samples yielded identical genotypes, and eight new genotypes were detected in the remaining samples. This analysis provided a minimum estimate of 16 mountain lions (seven identified by capture and nine identified by faecal DNA) living in or travelling through Yosemite Valley from March 1997 to August 1998. Match probabilities (probabilities that identical DNA genotypes would be drawn at random a second time from the population) indicated that the samples with identical genotypes probably came from the same mountain lion. Our results demonstrate that faecal DNA analysis is an effective method for detecting and identifying individual mountain lions.     

Predicting feed intake and feed efficiency in lactating dairy cows using digesta marker techniques

Direct measurement of individual animal dry matter intake (DMI) remains a fundamental challenge to assessing dairy feed efficiency (FE). Digesta marker, is currently the most used indirect technique for estimating DMI in production animals. In this meta-analysis we evaluated the performance of marker-based estimates against direct or observed measurements and developed equations for the prediction of FE (g energy-corrected milk (ECM)/kg DMI). Data were taken from 29 change-over studies consisting of 416 cow-within period observations. Most studies used more than one digesta marker. So, for each observed measurement of DMI, faecal dry matter output (FDMO) and apparent total tract dry matter digestibility (DMD), there was one or more corresponding marker estimate. There were 924, 409 and 846 observations for estimated FDMO (eFDMO), estimated apparent total tract DMD (eDMD) and estimated DMI (eDMI), respectively. The experimental diets were based mainly on grass silage, with soya bean or rapeseed meal as protein supplements and cereal grains or by-products as energy supplements. Across all diets, average forage to concentrate ratio on a dry matter (DM) basis was 59 : 41. Variance component and repeatability estimates of observed and marker estimations were determined using random factors in mixed procedures of SAS. Between-cow CV in observed FDMO, DMD and DMI was, 10.3, 1.69 and 8.04, respectively. Overall, the repeatability estimates of observed variables were greater than their corresponding marker-based estimates of repeatability. Regression of observed measurements on marker-based estimates gave good relationships (R²=0.87, 0.68, 0.74 and 0.74, relative prediction error =10.9%, 6.5%, 15.4% and 18.7%for FDMO, DMD, DMI and FE predictions, respectively). Despite this, the mean and slope biases were statistically significant (P<0.001) for all regressions. More than half of the errors in all regressions were due to mean and slope biases (52.4% 87.4%, 82.9% and 85.8% for FDMO, DMD, DMI and FE, respectively), whereas the contributions of random errors were small. Based on residual variance, the best model for predicting FE developed from the dataset was FE (g ECM/kg DMI)=1179(±54.1) +38.2(±2.05)×ECM(kg/day)−0.64(±0.051)×BW (kg)−75.6(±4.39)×eFDMO (kg/day). Although eDMD was positively related to FE, it only showed a tendency to reduce the residual variance. Despite inaccuracy in marker procedures, eFDMO from external markers provided a reliable determination for FE measurement. However, DMD estimated by internal markers did not improve prediction of FE, probably reflecting small variability.     

Evaluation of thermophilic anaerobic digestion processes for full-scale Class A biosolids disinfection at Hyperion Treatment Plant

This paper describes 5 phases of full-scale testing at the City of Los Angeles Hyperion Treatment Plant (HTP) for producing Class A biosolids (U.S. EPA Part 503 Biosolids Rule) by thermophilic anaerobic digestion. Phases I and II were tests with a two-stage continuous-batch process in a thermophilic battery of six digesters and a designated post-digestion train that was isolated from mesophilic operations. These tests demonstrated that digester outflow biosolids met the Class A limits for fecal coliforms and Salmonella sp. However, fecal coliform densities sharply increased during post-digestion. The recurrence was possibly related to a combination of a large drop of the biosolids temperature after the dewatering centrifuges and contamination of thermophilically digested biosolids from mesophilic operations. Phase III was conducted after insulation and electrical heat-tracing of the post-digestion train to maintain a biosolids temperature throughout post-digestion at about the same level as in the digester outflow. Biosolids monitoring at the last points of plant control (silos at Truck Loading Facility and farm for land application) indicated that fecal coliform recurrence was prevented. After completing the conversion of HTP to thermophilic operation, certification tests of Phases IV and V demonstrated Class A compliance of a two-stage continuous-batch process under Alternatives 1 and 3 of the Part 503 Biosolids Rule, respectively. HTP received the permit for Class A (indeed exceptional quality) biosolids land application in Kern County, California, in December 2002 under Alternative 3. Since 2003, HTP has consistently complied with the federal and local standards for Class A biosolids, indicating that Class A limits can be met under conditions less stringent than defined by the Alternative 1 time-temperature requirement for batch treatment.     

Detection of genetic diversity by pulsed-field gel electrophoresis among Escherichia coli O157 isolated from bovine faecal samples by immunomagnetic separation technique

AIMS: Escherichia coli O157 is considered to be one of most important human pathogens of animal origin which causes serious clinical complications. One of the most common methods to isolate E. coli O157 is the immunomagnetic separation (IMS) technique which employs specific antibodies coupled to magnetic beads to bind and extract cells from enrichment broths followed by plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite (CT-SMAC) plates. The aim of this study was to determine strain variation by pulsed-field gel electrophoresis (PFGE) among E. coli O157 on IMS/CT-SMAC plates. METHODS AND RESULTS: Every suspect colony of E. coli O157 was tested following isolation by the IMS/CT-SMAC technique. From 124 colonies detected; six XbaI-PFGE profiles were identified. CONCLUSIONS: Our results demonstrate that mixed populations of E. coli O157 with distinguishable PFGE profiles that are simultaneously present in bovine faeces can be isolated with IMS/CT-SMAC technique. SIGNIFICANCE AND IMPACT OF THE STUDY: If the aim of the study was to analyse diversity of PFGE profiles of E. coli O157 in a faecal sample following isolation by the IMS/CT-SMAC technique, at least five colonies per sample should be analysed to detect different PFGE subtypes if present.     

Molecularly assessed shifts of Bifidobacterium ssp. and less diverse microbial communities are characteristic of 5-year-old allergic children

The composition of intestinal microbiota and the Bifidobacterium group community in 20 allergic and 20 nonallergic 5-year-old children was visualized by PCR-denaturing gradient gel electrophoresis (DGGE). The number of dominant bands in the DGGE profiles was smaller in allergic children than in nonallergic children (P<0.001). Allergic children mainly formed a single group upon cluster analysis, whereas nonallergic children were divided between four different groups. In allergic children the Bifidobacterium adolescentis species prevailed, and in nonallergic children the Bifidobacterium catenulatum/pseudocatenulatum prevailed (P=0.01 and P=0.01, respectively). The less diverse composition of intestinal microbiota and prevalence of particular species of Bifidobacterium were characteristic of allergic children even at the age of 5 years.     

Relationships between the density of different indicator organisms on sheep and beef carcasses and in frozen beef and sheep meat

AIM: To describe the relationship between the concentration of different indicator bacteria in red meat. METHODS AND RESULTS: Enumeration data for aerobic plate count (APC), Enterobacteriaceae, coliforms and Escherichia coli biotype I were analysed from an Australia-wide survey of beef carcasses, sheep carcasses, frozen beef and frozen sheep meat. In all commodities, there was only low-to-moderate rank correlation (0.16-0.47) between concentration of APC and concentration of each Gram-negative indicator. Rank correlations between counts of Gram-negative indicators were much higher (0.47-0.92) especially when nondetections were excluded from analysis (0.78-0.94). Receiver-operator characteristics analysis showed that detection of coliforms can predict the presence of E. coli biotype I with almost 100% sensitivity but fails to predict absence in 2.7-8.5% of samples not containing E. coli biotype I. CONCLUSIONS: Enumeration of coliforms is a useful adjunct to enumeration of E. coli biotype I or Enterobacteriaceae in red meat. The density of coliforms or Enterobacteriaceae can be used to predict the presence or absence of E. coli biotype I, although when the latter is at low prevalence errors in positive test prediction can be large. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative basis is provided for comparing the concentration of different indicator bacteria measured in the production, regulation and trade of red meat.     

Inference about the Number of Resident Bacterial Strains when Sampling from Plate Colonies

This paper deals with making inferences about the number of resident bacterial strains in mice gut based on the number of strains of bacteria found in a random sample of colonies derived from plated faecal material. It is shown that for what seems to be a natural way to test the hypothesis that there is only one resident bacterial strain against the alternative that there is more than one resident strain the probability of a Type II error may be unacceptably large. It is also shown that given the sample contains only one bacterial strain the probability that the animal has only one resident strain converges to unity at a geometric rate as the sample size increases.