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W Gaastra

Publications and source records attributed to W Gaastra.

At least 55 records · Page 3Linked to original sources
PubMed1996

Epidemiological characteristics of bovine clinical mastitis caused by Staphylococcus aureus and Escherichia coli studied by DNA fingerprinting.

OBJECTIVE: To study the epidemiology of clinical mastitis caused by Escherichia coli and Staphylococcus aureus by differentiating isolates with DNA fingerprinting techniques, using polymerase chain reaction. DESIGN: Milk samples were collected from cases of clinical mastitis in dairy cows. Escherichia coli and S aureus isolates from these cases were compared within and between cows and herds. SAMPLE POPULATION: Seven dairy herds with an average bulk milk somatic cell count < 150,000/ml, and incidence of cows with clinical mastitis of > 25%/y. PROCEDURE: Chromosomal DNA was isolated from E coli and S aureus strains isolated from cases of clinical mastitis, and amplified by polymerase chain reaction, using enterobacterial repetitive intergenic consensus primers for E coli and a random amplified polymorphic DNA primer for S aureus. Escherichia coli and S aureus strains were identified and differentiated, using their DNA polymorphism pattern. RESULTS: Multiple E coli genotypes were found in each of the herds. Persistent infections with E coli were sporadic. Only a limited number of different S aureus genotypes was found in each of the herds studied. Recurrent cases of S aureus mastitis were found in 25% of quarters with clinical S aureus mastitis. Comparing S aureus isolates from different herds indicated that 1 S aureus genotype was most prevalent. CONCLUSIONS: Because different quarters were infected with different genotypes, it was concluded that E coli is an environmental pathogen, and does not generally spread from quarter to quarter. The hypothesis that S aureus mastitis is a contagious disease, spreading from infected to uninfected quarters, could not be rejected.

Animals↗
PubMed1995

Identification of Escherichia coli strains from cows with clinical mastitis by serotyping and DNA polymorphism patterns with REP and ERIC primers.

A number of Escherichia coli strains was isolated during a study of clinical mastitis on seven farms in the Netherlands. From these E. coli strains, 30 were characterised with regard to their serotype and their DNA polymorphism pattern with REP and ERIC primers. Special attention was given to recurrent E. coli mastitis in cows. The combination of serotype and DNA pattern observed, was used to study the epidemiology of clinical E. coli mastitis. The results demonstrated that the PCR reaction with the ERIC primers can be used for differentiation of E. coli strains. The DNA polymorphism patterns showed that E. coli strains isolated from cases of clinical mastitis have a great variability in genotype. More 3 than one case of clinical mastitis associated with E. coli during the same lactation period occurred infrequently. However when it took place, E. coli strains isolated from the separate episodes of inflammation, were in most instances of the same serotype and had the same DNA amplification pattern.

Animals↗
PubMed1995

Swine dysentery: more unknown than known.

Swine dysentery (SD) is an economically important disease. It is caused by the spirochete Serpulina hyodysenteriae. In order to minimize the economic damage of SD, several methods to control this disease are recommended. Whereas hygienic measures and use of antimicrobials are actually practised for prevention, detection and exclusion of carriers of S. hyodysenteriae and vaccination against the disease still needs further attention. The last two methods require reliable and sensitive diagnostic tests and understanding of the pathogenesis of and immune development against SD. At present the detection of all individual carriers of S. hyodysenteriae is not yet assured, since the tests for screening individual animals are not satisfactorily evaluated as far as sensitivity and/or specificity are concerned. Studies on the pathogenesis of SD have been performed to develop a vaccine. Besides hemolysin/cytotoxin production, no other virulence factors have been identified with certainty for S. hyodysenteriae. Recently however, further indications for a role of motility in the pathogenesis of SD have been obtained. In this manuscript we summarize the most relevant recent findings.

Animals↗
PubMed1995

Isolation and identification of fimbriae and toxin production by Escherichia coli strains from cows with clinical mastitis.

A total of 20 Escherichia coli strains isolated from cases of bovine mastitis were examined for fimbriae production, for the presence of genes coding for enterotoxins (LT and ST1), verotoxins (VT), and for the production of cytotoxic necrotizing factors (CNF1 and CNF2). Fimbriae could be isolated from four strains. The N-terminal amino acid sequence of the fimbriae from two strains, was determined. The two sequences were almost identical and homologous to that of the major subunit of E. coli F17 fimbriae. A DNA probe was derived from this N-terminal sequence and used as probe in hybridization experiments with chromosomal DNA of the 20 strains. To test if the strains contained genes that code for the F17 adhesin and the F17 major subunit, a Polymerase Chain Reaction (PCR) assay was performed with primers based on the nucleotide sequence of the genes. Eleven of 20 strains contained sequences that were homologous with sequences for the F17 fimbrial subunit and the F17 adhesin. Strains were tested directly for toxin production on Hela cells and by PCR for the presence of toxin genes. One of the twenty strains, produced a CNF toxin. No strains reacted positive in the PCR for LT, ST1 and verotoxin genes.

Adhesins, Escherichia coli↗
PubMed1994

Characterization of three putative Serpulina hyodysenteriae hemolysins.

Serpulina hyodysenteriae hemolysin is though to be an important virulence factor in swine dysentery. One gene, tlyA, previously called tly, encoding a hemolysin in S. hyodysenteriae strain B204 has been characterized (Muir et al. Infect Immun 1992; 60: 529-35). Two other genes of S. hyodysenteria strain B204, designated tlyB and tlyC, encoding hemolytic proteins in Escherichia coli strain DH5 alpha were cloned and sequenced. The tlyB and tlyC genes, when expressed in E. coli, encode heat-labile, protease-sensitive proteins which exhibit both hemolytic and cytotoxic activity in vitro. The calculated molecular weights of the tlyB and tlyC gene products are 93.3 kDa and 30.8 kDa, respectively. The tlyB gene product has sequence homology with the Clp proteins, whereas for the tlyC-encoded protein no homology with other protein sequences was observed. Southern hybridization showed that the tlyB and tlyC genes are present in a single copy on the chromosome of S. hyodysenteriae.

Amino Acid Sequence↗
PubMed1994

Regions of the CFA/I promoter involved in the activation by the transcriptional activator CfaD and repression by the histone-like protein H-NS.

Expression of CFA/I fimbriae of Escherichia coli requires the transcriptional activator CfaD. The mechanism by which CfaD activates the CFA/I promoter is to overcome the repression by H-NS, one of the histone-like proteins in E coli. This study addresses the question of which sequences in the promoter region of CFA/I interact with CfaD and H-NS. In order to determine this, deletion mutants of the CFA/I promoter were constructed and cloned upstream of the promoterless lacZ gene. The effect of CfaD and H-NS on the expression of these constructs was determined.

Antigens, Bacterial↗
PubMed1994

Genotypic and phenotypic identification of coli surface antigen 6-positive enterotoxigenic Escherichia coli.

A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.

Antigens, Bacterial↗
PubMed1993

Invasion of HeLa cells by Bordetella bronchiseptica.

Invasion, defined as adhesion to, followed by entrance into HeLa cells by Bordetella bronchiseptica was determined by (i) specific staining of intracellular bacteria and (ii) counting of viable intracellular bacteria after killing extracellular bacteria with colistin. It was demonstrated for the first time that B. bronchiseptica, like Bordetella pertussis and Bordetella parapertussis, is able to invade HeLa cells. Comparison of the invasiveness of Bvg+ and Bvg- B. bronchiseptica showed that B. bronchiseptica, in contrast to B. pertussis, invaded HeLa cells in both phases. The number of viable intracellular bacteria isolated after invasion of bacteria in the Bvg- (flagellated) phase was ten-fold lower than when Bvg+ (fimbriated) bacteria were used. Strains which are deficient in the production of either FimX or Fim2 fimbriae were as invasive as wild-type B. bronchiseptica, which indicates that not these fimbriae but probably filamentous haemagglutinin (FHA) is the major adhesin of B. bronchiseptica.

Bacterial Adhesion↗
PubMed1993

Identification of Bordetella avium using the polymerase chain reaction.

A DNA fragment from Bordetella pertussis, encoding the fim2 fimbrial subunit gene with adjacent sequences, was used as a probe for the detection of homologous sequences in chromosomal DNA of Bordetella avium. A 1.8 kb Sa1I-PstI fragment from the genome of B. avium, which hybridized with the probe, was isolated and sequenced. No fimbrial subunit gene was located on the B. avium DNA fragment. Two regions could be distinguished in the sequence of the fragment. Region 1, which was 80% identical to the sequence upstream of the fim2 gene of B. pertussis and region 2, which had no identity with any known sequence. A 491 bp EagI DNA fragment (probe A) within region 1 and a 650 bp EagI DNA fragment (probe B) within region 2 were used as DNA probes on restriction endonuclease digests of chromosomal DNA from various bacterial species. This hybridization experiment showed that the region 2 sequence was specific for B. avium. A polymerase chain reaction (PCR) with specific primers within region 2 resulted in the amplification of a 500 bp DNA fragment with B. avium DNA only. This PCR is a useful method for the rapid detection of B. avium and appeared useful to discriminate B. avium from other Bordetella species and also from Alcaligenes faecalis.

Alcaligenes↗
PubMed1993

Induction of colonization factor antigen I (CFA/I) and coli surface antigen 4 (CS4) of enterotoxigenic Escherichia coli: relevance for vaccine production.

Regulatory proteins control the expression of the fimbrial colonization factor antigens CFA/I and CS4 of enterotoxigenic Escherichia coli (ETEC). To examine the mechanism behind lack of expression of these antigens in spontaneous CFA-negative mutants, we mobilized a recombinant plasmid harbouring the cfaD gene, which encodes a positive regulator of CFA/I and CS4 expression, into such derivatives. In electron microscopy, the induced surface structures were morphologically identical to the fimbriae of the CFA/I+ and CS4+ wild type strains. Immunogold labelling with monoclonal antibodies showed that the distribution of CFA/I and CS4 specific epitopes along the induced fimbriae was indistinguishable from that of the wild type strains. The percentage of fimbriated cells was consistently higher in the cfaD transformants than in the corresponding wild type strains. The present work reports on the efficiency of the cloned cfaD gene in restoring and enhancing the production of morphologically intact CFA/I and CS4 fimbriae.

Antibodies, Bacterial↗
PubMed1993

The role of hemolysin(s) in the pathogenesis of Serpulina hyodysenteriae.

Serpulina (Treponema) hyodysenteriae, an anaerobic beta hemolytic spirochaete, is the etiologic agent of swine dysentery. Not much is known at present about the virulence factors of S. hyodysenteriae. However, the hemolysin production of this bacterium is generally accepted to be a virulence factor. To study the exact role of hemolysin production in the pathogenesis of swine dysentery, the gene encoding a hemolysin, tly, was cloned and its nucleotide sequence determined. After inactivation of this gene, the virulence of a tly-minus mutant in mice was tested. The mutant had reduced hemolysis indicating that the tly-encoded hemolysin was not the only hemolysin produced by S. hyodysenteriae. Mice infected with the tly-minus mutant had fewer cecal lesions than mice infected with the wild-type S. hyodysenteriae. It was concluded that the tly-encoded hemolysin might be an important virulence factor, but not the only one. Since it was demonstrated that spirochaetes can be transformed through electroporation, this has made a genetic approach to elucidate the pathogenesis of spirochaetal infections possible.

Animals↗
PubMed1993

Differential decay of RNA of the CFA/I fimbrial operon and control of relative gene expression.

CFA/I fimbriae on human enterotoxigenic Escherichia coli are composed of the CfaB protein, the product of the second gene of the CFA/I operon. We show here that CfaB is expressed at a higher level than other proteins of the CFA/I operon. mRNA encoding the CfaB protein is much more abundant than mRNA encoding CfaA, the first protein, together with CfaB or mRNA encoding CfaA only. Only one promoter, upstream of cfaA, is present. These data indicate that a primary transcript containing cfaA and cfaB is processed into a cfaA-specific mRNA and a cfaB-specific mRNA. The cfaA mRNA is unstable, while the cfaB mRNA is stable and therefore accumulates in CFA/I-producing E. coli. The cfaB mRNA is probably stabilized by a stem-loop structure downstream of the cfaB gene. No distinct mRNA fragments could be detected encoding the other two proteins, CfaC and CfaE, of the CFA/I operon. These results indicate that cfaC- and cfaE-specific mRNAs degrade very rapidly and/or are produced in small amounts.

Bacterial Proteins↗
PubMed1992

The positive regulator CfaD overcomes the repression mediated by histone-like protein H-NS (H1) in the CFA/I fimbrial operon of Escherichia coli.

CFA/I fimbriae of enterotoxigenic Escherichia coli are expressed at 37 degrees C and not at 20 degrees C. Expression of CFA/I fimbriae requires two DNA regions (regions 1 and 2) which are separated by 40 kb on the wild type plasmid. Region 2 encodes a protein (CfaD) which activates the promoter in region 1. We investigated whether the histone-like protein H-NS (H1) of E.coli is involved in the temperature regulated expression of CFA/I fimbriae. As demonstrated recently with other temperature regulated genes, a mutation in the gene coding for this nucleoid-associated H-NS (H1) protein resulted in derepression of CFA/I expression. CFA/I fimbriae were now expressed both at 20 degrees C and 37 degrees C. More strinkingly, the positive regulator CfaD was not needed for CFA/I expression in an H-NS- strain. This indicates that CfaD diminishes an inhibitory effect of the H-NS nucleoid-associated protein. We also showed that in the H-NS- strain the CfaD protein still has a positive effect on the transcription of CFA/I.

Antigens, Bacterial↗
PubMed1992

Nucleotide sequences of the major subunits of F9 and F12 fimbriae of uropathogenic Escherichia coli.

An Eco RV-Cla I fragment containing the gene encoding the F9 fimbrial subunit of the human uropathogenic Escherichia coli strain C1018 and a PstI-PstI fragment containing the F12 fimbrial subunit gene of the dog uropathogenic strain 1442 have been cloned and the nucleotide sequence of the fragments determined. The structural gene of the F9 fimbriae (FniA) codes for a protein of 165 amino acid residues with a signal peptide of 25 amino acids. The F12 fimbrial gene (FtwA) codes for a protein of 155 amino acids which is preceded by a single peptide of 21 amino acids. The amino acid sequences of the FniA and FtwA proteins deduced from the nucleotide sequence were compared with sequences of other known P-fimbrial subunit proteins. As expected, most differences between the various proteins were found in the hypervariable regions defined by van Die et al. (1987). The N-terminal sequence of the FtwA protein differs from the one published by Klemm et al. (1983). FtwA contains two deletions found in comparison to the other fimbrial subunits.

Amino Acid Sequence↗
PubMed1992

Characterization of Dutch porcine Serpulina (Treponema) isolates by restriction endonuclease analysis and DNA hybridization.

Genomes of 55 Dutch porcine Serpulina (Treponema) hyodysenteriae and non-pathogenic Serpulina isolates were characterized by restriction endonuclease analysis (REA) and DNA hybridization. The Dutch porcine isolates were compared with American Type Culture Collection (ATCC) strains of S. hyodysenteriae and S. innocens and isolates of S. hyodysenteriae with known serotypes (reference strains). REA of the Dutch S. hyodysenteriae isolates resulted in two main patterns, while the non-pathogenic isolates had many distinct REA patterns, all different from the S. hyodysenteriae strains. The S. hyodysenteriae reference strains all had distinct REA patterns, different from the Dutch strains. Upon Southern hybridization with a S. hyodysenteriae DNA fragment encoding a flagellar protein, all S. hyodysenteriae strains could be divided in two groups. The non-pathogenic Serpulina strains had many distinct hybridization patterns and hybridized less intensely. Upon hybridization with a S. hyodysenteriae DNA fragment encoding a haemolysin, DNA of all S. hyodysenteriae strains reacted in the same band. DNA of non-pathogenic Dutch Serpulina strains and S. innocens did not hybridize. It was concluded that there are two main genotypes of S. hyodysenteriae in the Netherlands. This could be of importance for recombinant DNA vaccine development.

Animals↗
PubMed1992

Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis.

A probe based on 16S ribosomal RNA (rRNA) sequences was developed to detect Mycobacterium paratuberculosis, the causative agent of Johne's disease in cattle. Three universal primers were used to sequence the amplified fragments of the 16S rRNA gene of various species of mycobacteria. When the nucleotide sequences were analysed, a deletion was detected in the sequence of the fast-growing species. An oligonucleotide probe (P) directed to this region was synthesised and hybridised directly with total RNA of various mycobacterial strains in a dot-spot assay. The probe detected M. paratuberculosis, some other slow-growing mycobacteria of the M. avium-intracellulare (MAI) complex, and one atypical strain, M. gordonae. To increase the sensitivity of the probe, a 413-bp fragment of the 16S rRNA gene of M. paratuberculosis between P and a second oligonucleotide primer was amplified and hybridised with a M. paratuberculosis/M. avium-specific probe. When faecal samples of cattle were tested, all culture-positive samples were positive in the PCR assay.

Animals↗
PubMed1992

Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue-specific adhesive properties.

Escherichia coli strains bind to Gal alpha 1-4Gal-containing glycolipids via P pili-associated G-adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G-alleles. We suggest that the Class I papG-allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G-adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual pap/prs genes have occurred. We propose that the retention and spread of pap/prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.

Adhesins, Escherichia coli↗