Biomedical subjects

B A van der Zeijst

Publications and source records attributed to B A van der Zeijst.

At least 19 recordsLinked to original sources

PubMed2000

DNA rearrangements in the flagellin locus of an flaA mutant of Campylobacter jejuni during colonization of chicken ceca.

Campylobacter jejuni is an enteropathogen for humans but commensal for chickens. In both hosts, the flagella and motility are important colonization factors. The flagellin gene is duplicated in Campylobacter, but only one flagellin gene, flaA, is sufficient for motility. We found that, during colonization of the chicken intestine, a nonmotile flaA mutant of C. jejuni underwent rearrangements within its flagellin locus, thereby regaining its motility and colonization capacity. In contrast, in vitro motile revertants isolated from liquid culture showed different flagellin DNA rearrangements than after reversion in the chicken.

Animals↗

PubMed1999

A serological study of cohorts of young dogs, naturally exposed to Ixodes ricinus ticks, indicates seasonal reinfection by Borrelia burgdorferi sensu lato.

Thirty-three family dogs were monitored for antibodies to Borrelia burgdorferi sensu lato over a 3-year period. Serum samples were collected before and during the season of high tick activity. Antibody levels were measured with an ELISA based on whole-cell antigens and an ELISA with a purified recombinant flagellin (r410). Antibody levels measured with the whole-cell ELISA increased after the first exposure to ticks. Following the first seasonal period of tick quiescence, antibody levels decreased, and subsequently increased again in the second tick season. Thereafter whole-cell ELISA titres persisted at moderate levels and did not decrease between tick seasons. The recombinant flagellin ELISA did not show a strong response in the first tick season, but did in the second tick season and levels of antibodies continued to fluctuate thereafter. We conclude that most dogs in this study developed an antibody response against Borrelia burgdorferi sensu lato after their first tick infestation and were thereafter repeatedly immunologically stimulated, probably reinfected, during the consecutive tick seasons.

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↗

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

Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae.

Streptococcus pneumoniae comprises 90 serotypes, each one having its own specific polysaccharide capsule. In order to explore the diversity of capsular polysaccharide synthesis (cps) gene clusters in S. pneumoniae, we performed cross-hybridizations between the 12 cps genes of S. pneumoniae serotype 14 and chromosomal DNA of 26 strains comprising 26 different capsule types. Large variations in the hybridization patterns were observed. The genes cps14A to cps14D are conserved in most serotypes. Sequences homologous to cps14I to cps14L were only observed in the four types of serogroup 15, which all have a capsule structure similar to that of type 14. By using a cps14E knock-out construct, cpsE mutants of the pneumococcal types 9N, 13, and 15B were obtained. These mutants were unencapsulated and showed reduced glycosyltransferase activity, indicating that the pneumococcal types 9N, 13, and 15B express a glucosyl-1-phosphate transferase which is homologous to Cps14E. Glycosyltransferase assays showed that among 21 pneumococcal types which contain glucose in the core of their capsule polysaccharide, 19 types express glucosyl-1-phosphate transferase activity. However, not all of these types hybridized strongly with Cps14E, the type 14 glucosyl-1-phosphate transferase gene. Thus, pneumococci possess glucosyltransferase genes distinct from cps14E, but encoding enzymes with identical activity. All serotypes which synthesized lipid-linked lactose intermediates in glycosyltransferase activity assays (type 11B, 13, 15F, 15A, 15B, 15C) hybridized with cps14G. This gene encodes a galactosyltransferase which catalyzes the addition of 1,4-linked beta-galactose to lipid-linked glucose. The cps14G homologues in type 11B, 13, 15F, 15A, 15B, and 15C may encode a similar beta-galactosyltransferase activity as cps14G in type 14.

Bacterial Capsules↗

PubMed1998

Identification of Campylobacter jejuni promoter sequences.

A promoterless lacZ shuttle vector, which allowed screening of promoters by beta-galactosidase activity in Campylobacter jejuni and Escherichia coli, was developed. Chromosomal DNA fragments from C. jejuni were cloned into this vector; 125 of 1,824 clones displayed promoter activity in C. jejuni. Eleven clones with strong promoter activity in C. jejuni were further characterized. Their nucleotide sequences were determined, and the transcriptional start sites of the putative promoters in C. jejuni were determined by primer extension. Only 6 of these 11 promoters were functional in E. coli. The 11 newly characterized and 10 previously characterized C. jejuni promoters were used to establish a consensus sequence for C. jejuni promoters. The 21 promoters were found to be very similar. They contain three conserved regions, located approximately 10, 16, and 35 bp upstream of the transcriptional start point. The -10 region resembles that of a typical sigma70 E. coli promoter, but the -35 region is completely different. In addition a -16 region typical for gram-positive bacteria was identified.

Base Sequence↗

PubMed1997

Cloning and characterization of the lytB gene of Campylobacter jejuni.

LytB of Escherichia coli is an essential gene involved in penicillin tolerance and the stringent response. The lytB gene of Campylobacter jejuni was cloned and characterized. It could complement a temperature-sensitive E. coli lytB mutant. The C. jejuni lytB gene encodes a protein of 277 amino acids that has 34, 36 and 40% amino acid identity with the LytB proteins of E. coli, Haemophilus influenzae, and Synechocystis sp. PCC6803, respectively. The lytB gene is situated between the aroA gene and a gene that encodes ribosomal protein S1.

Bacterial Proteins↗

PubMed1997

Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14.

Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.

Amino Acid Sequence↗

PubMed1997

Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene.

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene from C. Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification of C. jejuni, C. coli, C. lari and C. upsaliensis was developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp.

Amino Acid Sequence↗

PubMed1997

Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit.

We have reported previously on seven genes (cps14B-H) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115-bp-long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14A. All 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the beta-1,3-N-acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a beta-1,4-galactosyltransferase, which requires beta-linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.

Amino Acid Sequence↗

PubMed1996

The aroA gene of Campylobacter jejuni.

The gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aroA) cloned from Campylobacter jejuni (Cj) strain 81116 was identified by complementation of an Escherichia coli (Ec) auxotrophic aroA mutant. The Cj aroA gene has been sequenced. It encodes an enzyme of 428 amino acids (aa), that is homologous to other bacterial EPSP synthases, especially that of Bacillus subtilis with which it has a 39% aa identity. The transcriptional start point was mapped. It is present in an upstream open reading frame (ORF) that has a strong homology to the gene encoding phenylalanine tRNA synthetase (pheS). Downstream from aroA another ORF is present which is homologous to the lytB gene of Ec. The stop codon of the aroA gene overlaps the start codon of lytB.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Recombinant expression and use in serology of a specific fragment from the Cowdria ruminantium MAP1 protein.

The major antigenic protein (MAP1) of Cowdria ruminantium was screened for immunogenic regions by expression of overlapping recombinant DNA clones of the gene encoding the MAP1 protein. Two regions, designated MAP1-A and MAP1-B, were recognized by all antisera to 9 different isolates of C. ruminantium. MAP1-A contained one or more epitopes responsible for false-positive reactions with Ehrlichia antisera in several serological tests for cowdriosis. Cross-reactivity with MAP1-B was limited to antisera to Ehrlichia chaffeensis and Ehrlichia canis. Antisera to Ehrlichia species that infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not recognize MAP1-B. The sensitivity of an indirect ELISA based on MAP1-B was found to be excellent, since all sera from animals experimentally infected with C. ruminantium (64 out of 64) reacted with MAP1-B. Validation of this ELISA was carried out with field sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. Only 9 out of 111 samples from Zimbabwe, and 1 out of 58 samples from the Caribbean islands, which were considered to be false positives by immunoblot or indirect ELISA, reacted with MAP1-B. Thus, the ELISA based on MAP1-B is at present the most specific and sensitive serological test for cowdriosis.

Animals↗

PubMed1996

Differences between Taylorella equigenitalis strains in their invasion of and replication in cultured cells.

The ability of Taylorella equigenitalis, the causative agent of contagious equine metritis, to invade and replicate in equine derm cells was studied. The kinetics of invasion and replication were determined for four T. equigenitalis strains. On the basis of these experiments, a simpler assay in which the invasive as well as the replicative properties of a particular strain could be determined was developed. This assay was used to characterize 32 strains, which had previously been typed by field inversion gel electrophoresis of genomic restriction fragments. The invasiveness of T. equigenitalis strains ranged from 3 to 0.015 bacteria per cell and seemed to be associated with the contagiousness of the infection. The replication index (number of intracellular bacteria per cell at 24 h after inoculation divided by the number of intracellular bacteria per cell at 4 h after inoculation) varied from 1 to 857 and seemed to be associated with the severity of the symptoms of contagious equine metritis. There was no association between the invasiveness and the replication index of the strains, nor was there an association of invasion and replication with field inversion gel electrophoresis grouping.

Animals↗

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↗

PubMed1995

Analysis of flagellin gene expression in flagellar phase variants of Campylobacter jejuni 81116.

Flagella production in Campylobacter jejuni 81116 is subject to phase variation; the bacterium is able to switch its flagellum synthesis, and thereby its motility, on and off. Under standard laboratory growth conditions flagellar phase variants can be maintained as stable, pure cultures. We found conditions that efficiently induced a phase shift in vitro. The flaA gene but not the flaB gene is subject to the on and off switch. Minor amounts of FlaB are still present in aflagellate cells. We previously showed that flagellin gene expression in phase variants was regulated at the transcriptional level. Here, sequence data prove that abolishment of flaA transcription is not caused by DNA rearrangements or mutations within the flagellin locus. Since flaA is preceded by a typical sigma 28 promoter a C. jejuni sigma 28 homolog could play a role in regulation of flaA gene expression but such a gene or protein could not be detected. However, in vitro transcription could be detected using sigma 28-holoenzyme preparations from Bacillus subtilis. Possible regulatory mechanisms that may control flagellar phase variation in Campylobacter are discussed.

Bacterial Proteins↗

PubMed1995

Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer.

The capacity of Campylobacter jejuni to generate genetic diversity was determined for its flagellar region. Recombination within a genome, as well as recombination after the uptake of exogenous DNA, could be demonstrated. The subunit of the flagellar filament of C. jejuni is encoded by two tandem genes, flaA and flaB, which are highly similar and therefore subject to recombination. A spontaneous recombination within this locus was demonstrated in a bacterial clone containing an antibiotic-resistance gene inserted in flaA. A recombinant was isolated in which the antibiotic-resistance gene had been repositioned into flaB, indicating that genetic information can be exchanged between the two flagellin genes of C. jejuni. The occurrence of recombinational events after the uptake of exogenous DNA by naturally competent bacteria was demonstrated with two mutants containing different antibiotic-resistance markers in their flagellin genes. Double-resistant transformants were formed when purified chromosomal donor DNA was added to a recipient strain, when the two bacterial cultures were mixed under conditions that induce natural competence, or when the two strains were cocultured. Both mechanisms of recombination may be used by the pathogenic organism to escape the immunological responses of the host or otherwise adapt to the environment.

Base Sequence↗

PubMed1995

Identification of the domain which determines the g,m serotype of the flagellin of Salmonella enteritidis.

Clones expressing fragments of the flagellin protein of Salmonella enteritidis were constructed and screened with a g,m-specific monoclonal antibody. Results showed that the g,m epitope is localized between amino acids 258 and 348 of the flagellin. The fliC gene, encoding the flagellin of S. enteritidis, was proven to be the only flagellin gene present in S. enteritidis.

Amino Acid Sequence↗

PubMed1995

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B. In conclusion, recombinant MAP1-B may be a suitable antigen for a sensitive serological test for cowdriosis, with dramatically improved specificity.

Animals↗