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W Gaastra

Publications and source records attributed to W Gaastra.

At least 37 records · Page 2Linked to original sources
PubMed2000

Analysis of the F antigen-specific papA alleles of extraintestinal pathogenic Escherichia coli using a novel multiplex PCR-based assay.

Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic Escherichia coli, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of papA and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the papA alleles among 75 E. coli isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that papA sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of papA, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional pap fragments. Among the urosepsis isolates, the assay revealed considerable segregation of papA alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of papA alleles between lineages. Sequencing of papA from two strains that were papA positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel papA variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known papA alleles. These findings provide novel insights into the papA alleles of extraintestinal pathogenic E. coli and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of papA alleles available to any laboratory with PCR capability.

Alleles↗
PubMed1999

Occurrence and characterization of gastric Helicobacter spp. in naturally infected dogs.

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii-like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.

Animals↗
PubMed1999

Detection of spirochetes by polymerase chain reaction and its relation to the course of digital dermatitis after local antibiotic treatment in dairy cattle.

The aim of the study was to monitor the course of digital dermatitis after local antibiotic treatment in an experimental group (treated on diagnosis) and a control group (treated 5 days later). The present study was carried out on 2 farms involving 18 animals. Monitoring was performed by means of clinical findings and detection of spirochetes on the surface of the lesions, using a polymerase chain reaction. Superficial wound smears were taken before and after treatment. Twelve animals on both farms followed the classical healing process, but six animals responded poorly to treatment. We observed that without treatment, there was no self-cure in the control group within 5 days. There was a significant improvement in the clinical condition of all animals after treatment on both farms, during the follow-up period. The time until reappearance of new digital dermatitis lesions was not significantly different between the experimental and control group, but it was different between the two farms which could be due to the influence of farm factors. Using primers specific for Treponema denticola and Treponema vincentii, all the disease stages had at least one positive polymerase chain reaction result indicating the presence of spirochetes in samples of all the disease stages during the healing process. This implies that the spirochetes are not completely eradicated from the surface of the lesions after treatment. It was also observed that the classical ulcerative disease stage (M2) had relatively more positive polymerase chain reaction results compared to any other disease stage, showing a possible link between the presence of spirochetes and clinical disease.

Administration, Topical↗
PubMed1999

Recurrent clinical mastitis caused by Escherichia coli in dairy cows.

In this study, the occurrence of persistent intramammary infections caused by Escherichia coli with recurrent episodes of clinical mastitis caused by E. coli are described for a cohort of 300 Dutch dairy herds. Calculations on the recurrent episodes were based on data collected by dairy farmers. The genotype of the E. coli strains was determined by means of a polymerase chain reaction using enterobacterial repetitive intergenic consensus (ERIC) primers, resulting in a DNA fingerprint. Quarters in which the same E. coli genotype was found were considered to be persistently infected. In 4.77% of all episodes of clinical mastitis caused by E. coli, persistent intramammary infections caused by the same E. coli genotype were found. Based on the occurrence of the same genotypes, we concluded that, in 2.98% of all episodes, transmission of E. coli strains among quarters within one cow might have occurred. In 13.04% of all episodes of clinical mastitis caused by E. coli in the study, different E. coli genotypes were isolated from recurrent episodes of clinical mastitis within the same cow, indicating that these cows were highly susceptible to recurrent intramammary infections caused by E. coli.

Animals↗
PubMed1998

Cloning and characterization of the gene encoding the primary sigma-factor of Campylobacter jejuni.

The rpoD gene encoding the primary sigma-factor of Campylobacter jejuni was amplified from genomic DNA with degenerate oligonucleotide primers. The complete gene encodes a polypeptide of 622 amino acids and has a deduced M(r) of 72.6 kDa. This polypeptide is 40% identical to the RpoD (sigma 70) protein of Escherichia coli and has 66% identity with the Helicobacter pylori RpoD protein. A C. jejuni sigma 70 promoter, not recognized by the E. coli sigma 70 factor, could be activated in this bacterium in the presence of the cloned C. jejuni RpoD protein.

Amino Acid Sequence↗
PubMed1998

The efficacy of a whole cell pertussis vaccine and fimbriae against Bordetella pertussis and Bordetella parapertussis infections in a respiratory mouse model.

Due to local and systemic side-effects, the currently used, highly effective, whole-cell pertussis vaccines (WCVs) will be replaced by acellular vaccines (ACVs) in some countries. These ACVs contain detoxified pertussis toxin, either alone or in combination with the filamentous haemagglutinin, pertactin and fimbriae. Ongoing clinical trials show that ACVs are clearly less reactogenic than WCVs and that ACVs comprised of three to five proteins are highly efficacious in inducing protection against Bordetella pertussis infections. An important unresolved question is, what the effect will be of the switch from WCVs to ACVs on the incidence of Bordetella parapertussis infections, the second causative agent of pertussis. A comparison of the efficacy of WCVs and ACVs against B. parapertussis infection is required to answer this question. We show that the Dutch WCV, although prepared from B. pertussis strains, protects against B. parapertussis infection in a murine respiratory model, although less efficiently than against B. pertussis infection. It was shown previously that the ACV components pertussis toxin, FHA and pertactin did not protect against B. parapertussis infection in a murine respiratory model. We have investigated the efficacy of two other ACV components, B. pertussis serotype-2 and -3 fimbriae against B. parapertussis infection in the murine model. The B. pertussis fimbriae protected mice against B. parapertussis infection although less efficiently than against B. pertussis infection. This result indicates that B. pertussis and B. parapertussis fimbriae are antigenically distinct. B. pertussis fimbriae were found to be as efficacious as the WCV against B. pertussis infection. Our results are discussed in the light of the switch from WCVs to ACVs.

Animals↗
PubMed1998

Polymorphism in the Bordetella pertussis virulence factors P.69/pertactin and pertussis toxin in The Netherlands: temporal trends and evidence for vaccine-driven evolution.

The Bordetella pertussis proteins P.69 (also designated pertactin) and pertussis toxin are important virulence factors and have been shown to confer protective immunity in animals and humans. Both proteins are used in the new generation of acellular pertussis vaccines (ACVs), and it is therefore important to study the degree of antigenic variation in these proteins. Sequence analysis of the genes for P.69 and the pertussis toxin S1 subunit, using strains collected from Dutch patients in the period 1949 to 1996, revealed three P.69 and three S1 variants which show differences in amino acid sequence. Polymorphism in P.69 was confined to a region comprised of repeats and located proximal to the RGD motif involved in adherence to host tissues. Variation in S1 was observed in two regions previously identified as T-cell epitopes. P.69 and S1 variants, identical to those included in the Dutch whole-cell pertussis vaccine (WCV), were found in 100% of the strains from the 1950s, the period when the WCV was introduced in The Netherlands. However, nonvaccine types of P.69 and S1 gradually replaced the vaccine types in later years and were found in approximately 90% strains from 1990 to 1996. These results suggest that vaccination has selected for strains which are antigenically distinct from vaccine strains. Analysis of strains from vaccinated and nonvaccinated individuals indicated that the WCV protects better against strains with the vaccine type P.69 than against strains with non-vaccine types (P = 0.024). ACVs contain P.69 and S1 types which are found in only 10% of recent Dutch B. pertussis isolates, implying that they do not have an optimal composition. Our findings cast a new light on the reemergence of pertussis in highly vaccinated populations and may have major implications for the long-term efficacy of both WCVs and ACVs.

Amino Acid Sequence↗
PubMed1997

The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits.

The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes. The DNA sequences from both clones were nearly identical and contained four open reading frames. Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors. The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit. Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Antigens, Bacterial↗
PubMed1997

Expression of colonization factor antigen I fimbriae by enterotoxigenic Escherichia coli; influence of growth conditions and a recombinant positive regulatory gene.

Enterotoxigenic Escherichia coli (ETEC) may spontaneously lose the positive regulatory cfaR gene and thereby the capacity to express colonization factor antigen I (CFA/I). A recombinant plasmid harbouring the cfaR gene was transformed into cfaR-negative mutant ETEC strains. CFA/I expression of wild-type and cfaR-transformed ETEC cultivated in different liquid media was quantified. At 37 degrees C, a high level of CFA/I expression from wild-type and cfaR-transformed strains was observed after growth in CFA broth. Transformation enhanced CFA/I expression only marginally. The transformant cultures showed a considerable variation in CFA/I expression which was paralleled by the proportion of individual bacteria producing CFA/I. This heterogeneity could be explained by a variable tendency to structural CFA/I gene loss among individual cfaR-transformed bacteria.

Bacterial Proteins↗
PubMed1997

A new putative fimbrial colonization factor, CS19, of human enterotoxigenic Escherichia coli.

A gene probe derived from the colonization factor antigen I (CFA/I) operon cross-hybridized at very low stringency to plasmid DNA from coli surface antigen 17 (CS17)-producing enterotoxigenic Escherichia coli (ETEC) and from the ETEC strain F595C, which was negative for previously described CFAs, CSs, and putative colonization factors (PCFs). A 16-kDa protein was identified in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of heat extracts prepared after growth of strain F595C at 37 degrees C on CFA agar containing bile salts. Transmission electron microscopy revealed bile salt- and temperature-dependent expression of fimbriae with a diameter of 7 nm. After transformation with a recombinant plasmid harboring the cfaR gene, which encodes a positive regulator of several CFAs, PCFs, and CSs, the 16-kDa protein was hyperexpressed. Polyclonal antibodies raised against this protein bound to the fimbriae and inhibited the adhesion of F595C bacteria to tissue-cultured Caco-2 cells. Nucleotide sequence determination of the gene encoding the 16-kDa fimbrial subunit revealed a high degree of amino acid sequence homology to the CFA/I, CS1, CS2, CS4, CS14, and CS17 polypeptides. The term CS19 is proposed for the new fimbria.

Agglutination Tests↗
PubMed1997

Detection and characterization of the coli surface antigen 6 of enterotoxigenic Escherichia coli strains by using monoclonal antibodies.

We describe, for the first time, the production of monoclonal antibodies (MAbs) against coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) and their use for characterization and diagnosis of CS6. Two MAbs, MAbs CS6-20:11:9 and CS6-2A:14, were produced by immunizing mice with purified CS6 or CS6-containing bacterial extracts. The MAb specificity was demonstrated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunoelectron microscopy, which showed that the MAbs bound to CS6-expressing bacteria as well as to purified CS6 and CS6 structural subunits but not to CS6-negative bacteria or other purified ETEC colonization factors. By using bacterial recombinants, i.e., strains with a complete CS6 operon or parts thereof, it was found that both MAbs were specific for CssB, one of the two structural subunits of CS6. Although the MAbs bound specifically to the entire surface of CS6-expressing bacteria, no structure of CS6 could be identified. The usefulness of the MAbs for the detection of CS6 was evaluated in an inhibition ELISA and in a dot blot test. Ninety-two ETEC strains with known colonization factors were analyzed, and all CS6-positive strains were identified by either assay with MAb CS6-2A:14, whereas MAb CS6-20:11:9 failed to identify two CS6-positive strains; in no instance was any CS6-negative strain identified by either of the MAbs. Parallel analyses of 48 strains with a gene probe specific for the other structural subunit of CS6, i.e., CssA, and the MAb-based assays gave identical results, suggesting the simultaneous presence of both subunits.

Animals↗
PubMed1996

Genotyping by PCR, of Staphylococcus aureus strains, isolated from mammary glands of cows.

A total of 71 Staphylococcus aureus strains isolated from bovine mammary glands were identified and subtyped. The methods used to differentiate between the S. aureus isolates were the DNA polymorphism pattern after amplification with a Polymerase Chain Reaction using several primer combinations and phage typing. The DNA fingerprinting technique using RAPD, ERIC1R and ERIC primers proved to be useful in differentiating isolates of S. aureus. Differentiation of isolates using phage typing gave no additional information compared to the DNA technique. The outbreak of S. aureus in the herd studied was mainly caused by one S. aureus strain. Other strains were only found on three occasions, twice in subclinical infections and once from a case of clinical mastitis. In the latter case the dominant strain was isolated from a different quarter of the same cow. Four of the ten cows studied suffered from clinical mastitis. From those four cows, three remained infected with the same S. aureus strain despite antibiotic treatment.

Animals↗
PubMed1996

Isolation and characterisation of dog uropathogenic Proteus mirabilis strains.

Proteus mirabilis strains isolated from the urine of dogs with urinary tract infections, were characterised with respect to the production of haemolysin and fimbriae. In contrast to healthy dogs, P. mirabilis was also isolated in high numbers from the faeces of dogs suffering from recurrent urinary tract infections. Production of fimbriae was demonstrated by electron microscopy and the presence of genes for two different types of major fimbrial subunits (MR/P-like or UCA-like) was demonstrated by Southern hybridisation. These genes were absent in the Proteus vulgaris, Providentia rettgeri and Morganella morganii strains tested. All but one P. mirabilis strains were haemolytic and most strains produced fimbriae albeit in different amounts. The UCA fimbrial subunits from dog and human isolates have identical molecular weights and N-terminal sequences and are immunologically cross reactive. It was concluded that dog uropathogenic P. mirabilis strains are very similar to human uropathogenic P. mirabilis strains.

Amino Acid Sequence↗
PubMed1996

Colonization factors of human enterotoxigenic Escherichia coli (ETEC).

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of childhood and travellers' diarrhoea. The ability of ETEC to adhere to the intestinal epithelium of the host is an important virulence determinant, and adhesion is mediated by proteinaceous surface appendages called colonization factors.

Animals↗
PubMed1996

Colonization factors of enterotoxigenic Escherichia coli isolated from children in north India.

Colonization factor antigens (CFAs) mediate attachment of enterotoxigenic Escherichia coli (ETEC) to the intestinal mucosa and induce protective immunity against ETEC diarrhea. ETEC strains (n = 111) isolated from North Indian children from 1985 to 1989 were examined for CFAs and putative colonization factors (PCFs). CFA/IV was the most common factor (26%), followed by coli surface antigen 17 (CS17) (19%), CFA/I (14%), PCFO166 (7%), and CFA/II (5%), while 24% of the isolates were negative for CFAs and PCFs. Among the strains producing heat-stable and heat-labile toxin (ST+LT+ strains), the STaI gene was strongly associated with the absence of known CFAs and PCFs, making the STaI+LT+ isolates an interesting target for the identification of previously undescribed factors. Repetitive sequence--based polymerase chain reaction revealed that the CS17+ strains, although clonally related, represented endemically circulating strains with a diversity greater than that of the CFA/I+ strains, which showed a substantial clonal clustering.

Bacterial Proteins↗
PubMed1996

Characterization of the fim2 and fim3 fimbrial subunit genes of Bordetella bronchiseptica: roles of Fim2 and Fim3 fimbriae and flagella in adhesion.

With DNA probes derived from the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis, two homologous subunit genes of Bordetella bronchiseptica were identified and cloned. The nucleotide sequences of these genes were determined. Comparison of these nucleotide sequences with the B. pertussis fimbrial fim2 and fim3 subunit genes showed a pronounced homology. Therefore, the B. bronchiseptica genes were also designated fim2 and fim3. Expression of the two B. bronchiseptica genes was demonstrated by Western blotting (immunoblotting) with polyclonal antiserum directed against the Fim2 and Fim3 fimbrial subunit proteins of B. pertussis and by enzyme-linked immunosorbent assay with monoclonal antibodies. After growth of B. bronchiseptica in the presence of MgSO4, no expression of both fimbrial subunit genes was observed. While no fimbriae were expressed, expression of flagella was observed under these circumstances. A longer C-stretch (up to 19 cytosine residues) than the one in front of the fim2 and fim3 genes of B. pertussis is present in front of the B. bronchiseptica fimbrial genes. In adherence experiments, fimbriated (Bvg+) as well as flagellated (Bvg-) B. bronchiseptica bacteria were able to adhere to HeLa cells, whereas nonflagellated B. pertussis did not. This suggests that fimbriae as well as other factors (possibly flagella) contribute to adherence of B. bronchiseptica to eukaryotic cells.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of CS20, a new putative colonization factor of enterotoxigenic Escherichia coli.

An enterotoxigenic Escherichia coli (ETEC) strain producing a previously undescribed putative colonization factor was isolated from a child with diarrhea in India. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of bacterial heat extracts revealed a polypeptide band of 20.8 kDa when the bacteria were grown at 37 degrees C which was absent after growth at 22 degrees C. A specific rabbit antiserum raised against the purified 20.8-kDa protein bound specifically to the fimbriae, as shown by immunoelectron microscopy, and inhibited bacterial adhesion to tissue-cultured Caco-2 cells. Transformation with a recombinant plasmid harboring the cfaD gene, which encodes a positive regulator for several ETEC fimbriae, induced hyperexpression of the 20.8-kDa fimbrial subunit and a substantial increase in the proportion of bacterial cells that were fimbriated. The N-terminal amino acid sequence of the polypeptide showed 65 and 60% identity to the PCFO20 and 987P fimbriae of human and porcine ETEC, respectively. We propose the term CS20 for this new putative colonization factor of human ETEC.

Adhesins, Bacterial↗