Biomedical subjects

M Bülte

Publications and source records attributed to M Bülte.

13 recordsLinked to original sources

PubMed2004

[The logistic regression model including interactions between the factor variables demonstrated for the detection of E. coli O157.H7 in artificially contaminated minced beef].

Logistic regression is a powerful tool to analyse data sets with a dichotomous response variable. However, in most situations it is used as a model without interactions between the factor variables. This is done either by presumption or to avoid difficulties in the interpretation of the statistical results. In this article first the model of simple logistic regression without interactions is introduced followed by the expanded model with pairwise interactions between the factors. The application of both models is demonstrated at the present data set concerning the detection of E. coli O157.H7 in artificially contaminated minced beef. The influencing variables are the factors enrichment time, inoculation density, enrichment broth, subculturing medium, and state of samples (fresh vs. deep frozen). The statistical reanalysis displayed strongly differing results emphasizing the importance of interactions in logistic regression models. In particular, the odds ratio for E. coli detection dependant from the enrichment time (24 h vs. 6 h) (OR = 0.41) was strongly overestimated without simultaneous attention of the E. coli inoculation density (OR approximately equal to 0.2 to 0.02). In this context the possible interpretation of the interaction is discussed.

Animals↗

PubMed2004

[Occurrence of enterohaemorrhagic E. coli (EHEC) in domestic animals].

Among the verocytotoxin producing E. coli strains (VTEC) the enterohemorrhagic group (EHEC) have emerged as important source of serious disease in human, e.g. the haemolytic uremic syndrome (HUS). VTEC strains possess different virulence profiles where by virulence traits can be provided by the chromosome, by plasmids and, in the case of verocytotoxins (except: VT2e) by bacteriophages. The original and main reservoir are ruminants. In Germany, VTEC strains were isolated in ruminant stocks regularly. In part, the prevalence was estimated up to 100%. However, strains of important EHEC serovar groups, e.g. O157, O26, O111, O103 and O145 as main source of human infections are isolated rarly. This is even the case for food originated from those animals. The hygienic management to avoid fecal contamination of carcasses during the slaughter process is of crucial importance. Future preventive strategies in the field of primary production may be the development of vaccination programs and/or the feeding management to reduce the shedding of acid resistant VTEC. Slowly recognized environmental sources of infection and contamination are biotic (e.g. flys, rodents) and abiotic factors (e.g. pasture, water, feed). In an own study that investigated the prevalence of VTEC positive animals in free range cows during sojourn on pasture a significant increase was estimated. Even asymptomatic human carriers can serve as source of infection or contamination.

Animal Husbandry↗

PubMed2003

Toward an international standard for PCR-based detection of Escherichia coli O157. Part 1. Assay development and multi-center validation.

As part of a major European research project, a diagnostic PCR assay, including an internal amplification control, was developed and validated in a collaborative trial for the detection of Escherichia coli O157. The assay is based on amplification of sequences of the rfbE O157 gene. The collaborative trial, including 12 international laboratories, was carried out in two phases: phase (a) was performed with identical PCR reagents, including the internal control, provided by the sending laboratory; phase (b) was performed on the same samples and internal control but using in-house PCR reagents of own choice. Phase (a) showed an inclusivity (detection of target strains) of 96.8% and the exclusivity (negative response from nontarget strains) was 100%. The overall performance resulted of phase (a) in an accordance of 98.8, concordance of 98.6, and a concordance odds ratio of 1.11. Phase (b) results showed an accuracy of 100% with all partners and by using different polymerase types and thermocycler models. This indicates that the assay, under consideration as an international standard, was just as reproducible between laboratories, as repeatable within a laboratory. The assay is taken further for validation on carcass-rinse samples.

Animals↗

PubMed2002

Two methods for construction of internal amplification controls for the detection of Escherichia coli O157 by polymerase chain reaction.

For the detection of food born bacteria by polymerase chain reaction (PCR) in food products, an internal amplification control (IAC) is required in order to prevent false negative results that might be caused by PCR inhibitors. In the present study, two IACs were constructed using two different methods. These IACs were designed in a way that the same primer pair can be used to amplify the target DNA and coamplify the IAC. The first IAC with a size of approximately 200 bp was constructed by deleting a part of the amplicon of the original target DNA (500 bp) between the two primer sites to produce an IAC smaller than the target DNA. The second IAC with a size of approximately 600 bp was synthesized in a one step PCR reaction. The primers used in this reaction possessed 5' over-hanging ends, which were identical to the primers used in the diagnostic reaction, whereas their 3' ends were complementary to the (pUC19) predetermined DNA sequence of defined length and sequence. The concentration of IACs appeared to be critical. Too much IAC DNA template would out-compete the target DNA template, thus giving a false negative result. However the use of an optimal IAC concentration increased the reliability of the PCR assays and appeared to be useful for food diagnostics.

DNA Primers↗

PubMed2000

Occurrence of the vancomycin-resistant genes vanA, vanB, vanCl, vanC2 and vanC3 in Enterococcus strains isolated from poultry and pork.

It is suspected that the use of avoparcin as a feeding antibiotic for the fat stock contributes to development of cross-resistance against vancomycin and teicoplanin. After isolating enterococci strains from poultry and pork meat by cultivation on citrate azide Tween carbonate agar (CATC) and screening the vancomycin resistance on Columbia colistin nalidixic acid agar (CNA, supplemented with 5% sheepblood and 5 mg vancomycin/l) the polymerase chain reaction (PCR) was used for the detection of the vancomycin resistance genes vanA ('high level'), vanB ('moderate high level'), vanC1, vanC2 and vanC3 ('low level'). Out of 1643 E.-isolates from 115 poultry and 50 pork samples, 420 isolates could be identified as vancomycin resistant, 202 isolates of which carry the vanA, one isolate both the vanA and the vanC1, 38 isolates the vanC1, 14 isolates the vanC2, nine isolates both the vanC1 and the vanC3 gene and 156 isolates carry no gene. The vanB gene was not found in these isolates. Comparing vanA-positive food isolates with those from different human sources by means of the pulsed field gel electrophoresis (PFGE) it could clearly be demonstrated that they do not show homological fingerprints according to the source of origin. It is therefore unlikely that there is a close genetic relationship between isolates from animal foodstuff and humans.

Animals↗

PubMed2000

Investigation of human infections with verocytotoxin-producing strains of Escherichia coli (VTEC) belonging to serogroup O118 with evidence for zoonotic transmission.

Twenty verocytotoxigenic Escherichia coli (VTEC) O118 strains isolated between 1996 and 1998 from human patients in Germany were analysed for their serotypes, their virulence markers and their epidemiological relatedness. Three strains were typed as O118:H12, these carried only the VT2d-Ount variant gene and were not associated with diarrhoea or haemolytic uraemic syndrome (HUS). Seventeen strains were serotyped as O118:H16 or O118:non-motile (NM). These carried all the genes for VTI, eae and EHEC-haemolysin. The O118:H16/NM strains were from diarrhoea (13 cases) and HUS (2 cases). Sixteen of the patients were young infants and most infections were associated with a rural environment. Evidence for zoonotic transmission from cattle to humans was found in two cases. The epidemiological relationship between the human and bovine O118:H16/NM isolates was indicated by homogeneous plasmid patterns and by very similar XbaI restriction patterns obtained by pulsed-field gel electrophoresis. VTEC O118:H16/NM are emerging pathogens in Germany and should be classified as new enterohaemorrhagic E. coli (EHEC) types.

Adolescent↗

PubMed2000

Identification of central nervous system tissue in retail meat products.

A procedure to detect tissues from the central nervous system that involved quantification of cholesterol and immunochemical detection of neuron-specific enolase and glial fibrillary acidic protein was used to analyze 402 samples of heat-treated meat products from various food outlets in Germany. The cholesterol content of 16 samples (4.0%) indicated the possible presence of central nervous system tissue because the levels exceeded the normal maximum cholesterol content of cooked sausages. In 7 of these 16 heat-treated meat products, immunoblotting of both neuron-specific enolase and glial fibrillary acidic protein confirmed the presence of CNS tissue. Repeated sampling by veterinary officials and analysis by both cholesterol quantification and immunoblotting confirmed these findings. Whereas all of the control samples (with and without added central nervous system tissue) were correctly classified by both cholesterol quantification and immunoblotting, negative results of immunoblotting must be carefully interpreted in the case of intensively heat-treated meat products. Thus, studies have yet to establish an increase in sensitivity of immunoblotting of neuron-specific enolase and glial fibrillary acidic protein. However, the detection of illegal use of central nervous system tissue in heat-treated retail meat products demonstrates the need for suitable analytical methods to control transmissible encephalopathies and to enforce labeling laws.

Animals↗

PubMed1999

Development of an integrated procedure for the detection of central nervous tissue in meat products using cholesterol and neuron-specific enolase as markers.

The emergence of a new variant of Creutzfeldt-Jakob disease during the bovine spongiform encephalopathy epidemic has focused attention on the use of tissue from the central nervous system (CNS) in food. So far, the banning of CNS tissue could not be effectively controlled because procedures for detection were missing. With regard to preventive health protection and labeling law enforcement, we have developed an integrated procedure for the detection of CNS tissue in meat products. Herein, we show that antigenic characteristics of neuron-specific enolase (NSE) quantitatively survive technological treatment including severe homogenization and pressure heating. Using both poly- and monoclonal antibodies against NSE in the Western blot, bovine and porcine brain could be detected in sausages, albeit with varying sensitivity (1 to 4%). Sensitivity was increased after reduction of fat content (30 to 40%) of the samples by means of a soxhlet extraction. This made possible the detection of brain addition as low as 0.25% when using monoclonal antibodies. Immunohistology showed distribution of CNS tissue in heat-treated meat products to be homogeneous. Immunoreaction was not found to be bound to morphologically intact histological or cytological structures; however, it proved to be highly specific. The quantification of cholesterol provides a low-cost screening method for the rapid identification of meat products, suspicious with regard to CNS tissue addition. Cholesterol content increased by 26 mg per 100 g of fresh substance for each percentage of brain added to internally produced reference material. Using three different approaches (internal reference material, raw material, and field samples), a provisional cutoff point of normal cholesterol content was calculated for emulsion-type cooked sausages to be 115 mg/100 g (P < 0.05).

Animals↗

PubMed1995

The use of a PCR-generated invA probe for the detection of Salmonella spp. in artificially and naturally contaminated foods.

Part of the invasion A gene (invA) of slamonellae (Rahn et al., 1992) was amplified and labelled simultaneously with digoxigenin by the polymerase chain reaction (PCR). This was used as gene probe for a colony hybridization assay which included nitrocellulose filter incubation on modified Rambach agar. 312 Salmonella and 268 non-Salmonella strains were hybridized with the invA probe. No false-negative or false-positive results were obtained. In 11 beef samples, which had been contaminated artificially with Salmonella, the test strain was recovered quantitatively with the invA probe. Salmonellae could be detected in 29 samples of 104 further foods of animal origin by means of the gene probe assay in contrast to 27 samples which were positive by the standard method. The invA probe assay allows for the quantitative estimation of Salmonella in fresh meat samples within 48 h. However, with frozen samples a pre-enrichment step is necessary.

Bacterial Proteins↗

PubMed1990

[Detection of verotoxin-producing E. coli (VTEC) in healthy cattle and swine with the DNA-DNA colony hybridization method].

With the DNA-DNA colony hybridization technique using specific gene probes for Verotoxin 1 (VT 1) and Verotoxin 2 (VT 2) 2100 E. coli strains from healthy animals were tested. Ten out of 82 milk cows (21.2%), 20 out of 212 beef cattle (9.4%) and five out of 75 pigs (6.7%) were found to carry genes for VT 1, VT 2 or both toxins, respectively. Among these strains the biotypes 5 and 6 were predominant. Some of the serotyped isolates have been described to be pathogenic for humans, like O157:H7, 082:H8, 0116, 0113, 0126 and 091, respectively. The unexpected high incidence of VTEC positive healthy animals possibly indicates a health hazard for human beings. Further investigations on the incidence of VTEC in food are necessary.

Animals↗

PubMed1990

Detection and characterization of fecal verotoxin-producing Escherichia coli from healthy cattle.

Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1- and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e., O22, O39, O82, O91, O113, O116, O126, and O136. Two isolates from different bulls were identified as serotype O157:H7. Results obtained in this study indicate that cattle may be an important source of verotoxigenic E. coli involved in human disease.

Animals↗

PubMed1989

[Glucuronidase detection and indol capillary test as reliable rapid identification procedures for the detection of E. coli in foods--toxinogenic strains included].

The fluorogenic beta-D-Glucuronidase test, together with an Indol-capillary test for rapid identification of E. coli, were proved with 60 toxinogenic, 335 nontoxinogenic wild-type strains, and 87 other gram-negative isolates from food. With a sensitivity of 96.5% and a specificity of 95.6%, the fluorogenic assay can be recommended as a reliable method for presumptive determination of E. coli. For confirmation, a time-, material- and labour-saving Indol-capillary test with simultaneous demarcation of fluorescence-positive Salmonella spp. should be carried out. This includes enterotoxigenic E. coli as well, with the exception of some fluorescence-negative Verotoxin-producing strains. As the primary cultivation medium the Plate-count-Monensin-KCl-Agar supplemented with 50 micrograms/ml 4-methyl-umbelliferyl-beta-D-glucuronide (PMK-MUG) can be recommended. Not suitable appear such media containing lactose due to acidification, while metabolizing the carbohydrate. This may significantly reduce or extinguish the fluorescence. The procedure recommended here permits also a reliable determination of lactose-negative E. coli-biotypes.

Bacteriological Techniques↗

PubMedDate not supplied

[Detection and characterization of verocytoxin-producing Escherichia coli types (VTEC) from different sources].

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic E. coli (EHEC) belonging to a few serovars embracing strains of O26, O103, O111, O118, O145 and O157 serogroup, respectively. In own investigations 3.791 food specimen of animal origin were investigated by use of an enzyme-immuno-assay (EIA) and the polymerase chain reaction (PCR). All E. coli isolates (n = 459) of food as well as isolates from cattle feces (n = 440), from HUS patients (n = 50) and asymptomatic human carriers (n = 16) were investigated by means of the PCR using primer pairs for verocytotoxin genes (vtx1, vtx2, vtx2c, vtx2d, vtx2e), the E. coli attaching und effacing gene (eae), the enterohemolysin-gene (ehlyA) and the vt2 transporter protein-gene (ile X tRNA). Differences were found in respect to the eae- and the ile X tRNA genes, which could be detected in significantly higher ratios in the isolates from patients and human carriers. Furthermore vtx2d strains were exclusively analyzed to each 25% in food and cattle strains. In six food samples pathogenic strains of serovar O157 were detected whereas some of the cattle strains were estimated to belong to EHEC serovars O26:H11, O118:H- and O157:H7. The own data support the thesis that the risk for human beings is affiliated to a great extent by direct contact with ruminants, followed by person-to-person transmission. Regarding the epidemiological data the thesis that each VTEC strain ist potentially an EHEC strain can not longer be substantiated.

Animals↗