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M A Smits

Publications and source records attributed to M A Smits.

At least 55 records · Page 3Linked to original sources
PubMed1993

Recognition of Mycoplasma canis as part of the mycoplasmal flora of the bovine respiratory tract.

Mycoplasma strain C3b was isolated in the Netherlands from the lung of a pneumonic calf. Forty similar strains were isolated afterwards from calves in 19 other herds in different parts of the Netherlands. Eight strains from eight different herds were investigated in this study. Results of tests to determine whether the organism catabolized glucose were inconclusive. Four strains, including strain C3b, apparently catabolized glucose under some test conditions; the remaining four strains did not. Although strain C3b and similar strains were slightly different from canine M. canis strains in growth inhibition tests and glucose metabolism tests, we concluded that strain C3b and similar strains have to be classified as M. canis. A close contact between calves and dogs was observed in several herds where strain C3b or similar strains were isolated. This is the first report of M. canis isolated from cattle.

Animals↗
PubMed1993

Cloning and characterization of the Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) gene.

To study the role of Actinobacillus pleuropneumoniae-RTX-toxin III (ApxIII) in the pathogenesis of porcine pleuropneumonia, we cloned and characterized the gene encoding this toxin. For that purpose, we screened an expression library of genomic DNA of serotype 8 with an ApxIII-specific monoclonal antibody and isolated a 425-bp fragment of an immunoreactive clone. Using this fragment as a probe, we identified and cloned an overlapping chromosomal NsiI restriction fragment of 5.0 kbp. Escherichia coli cells that contained this fragment produced a protein similar to ApxIII. Like ApxIII, the protein had a molecular mass of approximately 120 kDa, was recognized by an ApxIII-specific antibody, killed porcine lung macrophages, and was not lytic for sheep erythrocytes. We concluded from these data that the 5.0-kbp NsiI fragment contained the ApxIII-coding gene. Nucleotide sequence analysis of the 5.0-kbp NsiI fragment revealed the presence of two genes, apxIIIC and apxIIIA. These genes coded for proteins ApxIIIC and ApxIIIA, respectively, which were 53 and 50% identical to the prototypic RTX proteins HlyC and HlyA of E. coli. We assumed that the apxIIIA gene coded for the structural RTX toxin and that the apxIIIC gene coded for its activator. In addition, we found that ApxIII could be secreted from E. coli by the heterologous RTX transporter proteins HlyB and HlyD. The deduced amino acid sequence of ApxIIIA was 50% identical to that of ApxIA and 41% identical to that of ApxIIA. We concluded that, beside ApxI and ApxII, ApxIII is the third RTX toxin produced by A. pleuropneumoniae.

Actinobacillus pleuropneumoniae↗
PubMed1993

Repeats in an extracellular protein of weakly pathogenic strains of Streptococcus suis type 2 are absent in pathogenic strains.

Streptococcus suis type 2 strains that are pathogenic for pigs produce a 110-kDa extracellular protein factor (EF). Nonpathogenic and weakly pathogenic strains do not produce EF or produce a protein (EF*) that is immunologically related to EF. To study the pathogenesis of S. suis type 2 in pigs and to develop tools and methods for the control of S. suis type 2 infections, we cloned and characterized the genes encoding EF and various EF* proteins. Analysis of the deduced amino acid sequences showed that the first 833 amino acids at the N terminus of the EF and EF* proteins were nearly identical. The proteins differed, however, at their C termini. Unlike the 110-kDa EF protein, the EF* proteins contained several repeated units of 76 amino acids. The number and arrangement of the repeats in the EF* proteins varied. The data suggest that the gene encoding EF could have evolved from an epf* gene by a specific deletion event. The lack of repeated amino acid units in the EF protein may be related to virulence.

Amino Acid Sequence↗
PubMed1993

Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed.

Actinobacillus pleuropneumoniae↗
PubMed1992

Influence of Actinobacillus pleuropneumoniae serotype 2 and its cytolysins on porcine neutrophil chemiluminescence.

The effects of Actinobacillus pleuropneumoniae serotype 2 and its metabolites on the oxidative activity of porcine neutrophils were studied by using a chemiluminescence technique. Viable A. pleuropneumoniae stimulated the production of oxygen radicals by neutrophils. After having reached a peak value, the oxidative activity decreased until a total inhibition of the oxidative activity of the neutrophils was achieved. All effects were neutralized with homologous convalescent-phase pig sera which had been adsorbed by heat-inactivated A. pleuropneumoniae. Inactivated bacteria and bacteria in the presence of chloramphenicol each had no influence on the oxidative activity of neutrophils. In contrast, a heat-labile factor in A. pleuropneumoniae culture supernatants stimulated and inhibited the oxidative activity of the neutrophils in a dose-dependent manner. Undiluted and low dilutions of culture supernatants were toxic for the phagocytes, while high dilutions stimulated the oxygen radical production of the neutrophils. These effects were neutralized with homologous convalescent-phase pig sera. In order to investigate whether the heat-labile factors in the culture supernatant could be cytolysins, we repeated the experiments with cytolysin II and cytolysin III produced by recombinant Escherichia coli. It was demonstrated that stimulation and inhibition could be reproduced by both cytolysins. In conclusion, the observations made in this study showed that A. pleuropneumoniae secretes heat-labile metabolites that stimulate neutrophil-oxidative metabolism at relatively low concentrations and kill the neutrophils at higher concentrations. Cytolysins may be responsible, at least in part, for these effects.

Actinobacillus pleuropneumoniae↗
PubMed1992

Comparison of the cytolysin II genetic determinants of Actinobacillus pleuropneumoniae serotypes.

Cytolysins (Cly) I, II, and III are toxins secreted by Actinobacillus pleuropneumoniae. These toxins are thought to play an important role in the pathogenesis of porcine pleuropneumonia. ClyI and ClyII are RTX toxins and in general these toxins are encoded by operons consisting of four genes, C, A, B, and D. Our group recently cloned the C and A genes of the ClyII operon (clyIICA) of serotype 9. We found that this ClyII operon is truncated and lacked intact B and D genes (clyIIBD). B and D genes of the ClyI operon (clyIBD) were present however in serotype 9. In this study we analyzed the ClyII operons of the reference strains of the 12 A. pleuropneumoniae serotypes and compared them with the ClyII operon of serotype 9. We focused on (i) the presence, (ii) the sequence similarity, and (iii) the genomic environment of the clyIICA genes. The presence of the clyIICA sequences was studied by hybridization analysis of genomic DNA. The sequence similarity was studied by restriction fragment analysis on polymerase chain reaction-amplified DNA. The genomic environment was compared by analysis of the sequences that are located 3' of the clyIICA genes. We demonstrated that the clyIICA genes (i) are present in the reference strains of all serotypes, except serotype 10, (ii) have a high degree of sequence similarity, and (iii) are not contiguous with intact clyIIBD genes. We conclude that the organization and nucleotide sequence of the ClyII operons of A. pleuropneumoniae are very similar. We also studied the presence of clyIBD sequences and found them to be present in the reference strains of all serotypes, except serotypes 3 and 6. Thus, in most serotypes the clyIBD genes may complement the absent clyIIBD genes.

Actinobacillus pleuropneumoniae↗
PubMed1992

Cloning and nucleotide sequence of the gene encoding the 136-kilodalton surface protein (muramidase-released protein) of Streptococcus suis type 2.

We cloned and sequenced the gene encoding the muramidase-released protein (MRP) of a pathogenic Streptococcus suis type 2 strain to determine whether its amino acid sequence resembles that of proteins with known functions and to determine its function in virulence. The complete nucleotide sequence composing the gene and the regions flanking it was determined. The deduced amino acid sequence revealed the presence of a signal peptide at the N terminus and a cell envelope anchor at the C terminus, both of which resembled similar regions in several other surface proteins from gram-positive bacteria. The processed form of MRP has a length of 1,209 amino acids and a calculated molecular weight of 131,094. A highly repetitive region preceded the envelope anchor. The repeated units were preceded by a proline-rich stretch of amino acids (26 of 86). No overall homologies were observed between the amino acid sequence of MRP and protein sequences in the EMBL data bank. A particular region within the amino acid sequence, however, showed some similarity with the fibronectin-binding protein of Staphylococcus aureus. Binding of MRP to human fibronectin, however, could not be confirmed.

Amino Acid Sequence↗
PubMed1991

Cytolysins of Actinobacillus pleuropneumoniae serotype 9.

Cytolysin I (ClyI) and cytolysin II (ClyII), which are present in the culture supernatant of Actinobacillus pleuropneumoniae serotype 9, are thought to play an important role in the pathogenesis of pig pleuropneumonia. The purpose of this study was to clone and characterize the genetic determinants of these cytolysins. Cloning was accomplished by the screening of DNA libraries for the presence of cytolytic activity and for the presence of DNA sequences homologous to leukotoxin DNA of Pasteurella haemolytica. Both genetic determinants were found to be members of the RTX cytotoxin family. The ClyII determinant was characterized in more detail. It appeared that ClyII more closely resembled the leukotoxin of P. haemolytica than the alpha-hemolysin of Escherichia coli. The ClyII amino acid sequence was identical to a hemolysin gene sequence of A. pleuropneumoniae serotype 5; this finding indicates that the latter gene also codes for ClyII and not for ClyI, as has previously been suggested. The genetic organization of the ClyII determinant differed from the genetic organization of other RTX determinants. Genes responsible for secretion of ClyII were not contiguous with the toxin gene. Instead, secretion genes were present elsewhere in the genome. These secretion genes, however, belong to the ClyI operon. This indicates that the secretion genes of the ClyI operon are responsible for secretion of ClyI and ClyII.

Actinobacillus pleuropneumoniae↗
PubMed1991

Identification of hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae by use of monoclonal antibodies.

Monoclonal antibodies (MAbs) were raised against extracellular hemolytic and cytotoxic proteins of Actinobacillus pleuropneumoniae serotypes 2 and 9. MAbs were tested for inhibition of hemolytic and cytotoxic activities of the reference strains of A. pleuropneumoniae serotypes 1 to 12. Five MAbs inhibited hemolytic or cytotoxic activities of one or more serotypes and were used to probe Western blots (immunoblots) of cytotoxic and hemolytic culture filtrates of serotypes 1 to 12. The MAbs recognized three different proteins: (i) a protein of approximately 103 kDa that was associated with a weak hemolytic activity and a moderate cytotoxic activity, (ii) a protein of approximately 105 kDa that was associated with a strong hemolytic activity and a strong cytotoxic activity, and (iii) a protein of approximately 120 kDa that was associated with a strong cytotoxic activity but not with hemolytic activity. Serotypes 6, 7, and 12 produced only the 103-kDa protein, and serotype 10 produced only the 105-kDa protein. The other serotypes produced two proteins: serotypes 1, 5, 9, and 11 produced the 103- and 105-kDa proteins, and serotypes 2, 3, 4, and 8 produced the 103- and 120-kDa proteins.

Actinobacillus↗
PubMed1991

Sample preparation method for polymerase chain reaction-based semiquantitative detection of Leptospira interrogans serovar hardjo subtype hardjobovis in bovine urine.

An improved method of preparing bovine urine samples was developed for the rapid, specific, and sensitive detection of Leptospira interrogans serovar hardjo (subtype hardjobovis) DNA by the polymerase chain reaction (PCR). A total of 100 leptospire-free cows, 4 experimentally infected cows, and 2 negative control cows were used. PCR results were improved by (i) using 10-ml urine samples instead of 1-ml samples, (ii) adding 10(7) to 10(8) Leptospira biflexa serovar patoc cells as a carrier to each treated sample, (iii) preventing the loss of pelleted leptospires, and (iv) minimizing the presence of PCR-inhibiting factors in the samples. The preparation method enabled us to use the PCR to reproducibly detect as few as 5 to 10 leptospires per ml of urine without the need for dot blot hybridization. In addition, we were able to estimate the number of leptospires shed by experimentally infected cows.

Animals↗
PubMed1990

Cloning and expression of the dermonecrotic toxin gene of Pasteurella multocida ssp. multocida in Escherichia coli.

A DNA library of Pasteurella multocida ssp. multocida strain CVI 47459 was constructed in the Lambda GEM-11 vector. Recombinant clones that encoded dermonecrotic toxin (DNT) were identified immunologically with antiserum raised against purified DNT. By comparing the DNA restriction maps of the immunoreactive recombinants, we located the DNT gene. Hybridization studies with 10 strains of P. multocida ssp. multocida suggested that strains that do not produce the DNT do not contain sequences homologous to the DNT gene.

Bacterial Toxins↗
PubMed1990

Specificity of DNA probes for the detection of toxigenic Pasteurella multocida subsp. multocida strains.

Five DNA probes directed against different regions of the gene that encodes the dermonecrotic toxin of Pasteurella multocida subsp. multocida were examined for their ability to identify toxigenic P. multocida subsp. multocida strains. The specificities of the probes were studied with 96 strains of P. multocida subsp. multocida and 22 strains of 11 other bacterial species. Results of colony hybridization assays using these probes indicated that two of the five probes have potential diagnostic value.

Animals↗
PubMed1989

Nucleotide sequence and expression of a beta-tubulin gene from Plasmodium falciparum, a malarial parasite of man.

Genomic and cDNA clones, containing a Plasmodium falciparum beta-tubulin coding sequence (pf-bTub), were isolated and characterized. Comparison of the genomic sequence with the cDNA sequence showed that the malarial bTub-coding region is interrupted by two introns, the positions of which are not found in any beta-tubulin gene (btub) from other species. The gene appears to be present as a single-copy gene in the P. falciparum genome and is expressed as a 2.3-kb transcript both in the asexual blood stages and in the sexual stages (gametes/zygotes) of the parasite. The deduced polypeptide product of the pf-btub gene is a protein of 445 amino acids (aa) (Mr 49,517). Comparison of the aa sequence of pf-bTub with that of bTubs from other species revealed that the malarial protein shows a high degree of similarity to mammalian bTubs. Upon examination of the colchicine-binding sites of pf-bTub we predict that this tubulin probably has an altered sensitivity to this inhibitor.

Amino Acid Sequence↗
PubMed1989

Stage-specific expression and genomic organization of the actin genes of the malaria parasite Plasmodium falciparum.

Two different actin transcripts are found in the human malaria parasite Plasmodium falciparum. One of these is a 2.5-kb-long RNA found both in asexual blood stages and in the sexual stages (i.e., gametes/zygotes) of the parasite. This transcript is encoded by the P. falciparum (pf)-actin I gene. The second malarial actin gene, the pf-actin II gene, yields a 1.9-kb-long transcript which is formed solely in the sexual stages. Elucidation of the genomic organisation of these two Plasmodium actin genes showed that the pf-actin I gene does not possess any introns whereas the coding region of the pf-actin II gene is interrupted by a 368-bp intron. This intron has consensus splice junction sequences. Nucleotide sequence analysis of the 3' non-coding regions of the pf-actin genes revealed that these regions are quite long (pf-actin I, 250 bp; pf-actin II, 331 bp) and that these trailers do not share sequence similarity. Furthermore, the poly(A)+ addition sites of both actin mRNAs have now been identified. The 5' untranslated regions are also rather long; the sequenced areas lack sequence similarity and have, as do the 3' untranslated regions, a very high A + T content.

Actins↗
PubMed1989

Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum.

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.

Acylation↗
PubMed1988

Nucleotide sequence and deduced amino acid sequence of a Plasmodium falciparum actin gene.

The nucleotide sequence of a Plasmodium falciparum actin gene has been established. The gene codes for a protein of 376 amino acids and is not interrupted by introns. The nucleotide sequence reveals an extreme bias in codon usage. Not less than 85% of the codons possess an A or T at the third position. As has been found for the actins in other unicellular eukaryotes, P. falciparum actin is related both to vertebrate cytoplasmic and vertebrate muscle specific actins. However, the malarial actin is one of the most alpha-like actins hitherto found in lower eukaryotes.

Actins↗
PubMed1988

Extremely diverged actin proteins in Plasmodium falciparum.

In a previous paper the nucleotide sequence of a complementary DNA coding for a Plasmodium falciparum actin protein (pf-actin I) has been described. Here we present evidence that the genome of this human malaria parasite encodes for still another actin protein (pf-actin II). Via nucleotide sequence analysis of its coding DNA we established the amino acid sequence of this protein. This sequence was compared with the pf-actin I sequence and those of a number of other actins. The comparative studies revealed that the amino acid sequence of pf-actin II is very diverged from the actins known thus far. The mutual amino acid sequence similarity between both Plasmodium actins is also very poor and in fact the observed value is the lowest ever seen between actins within one species. Furthermore, the studies suggest that the actin genes from sporozoa and ciliated protozoa, but not those from amoebae, have evolved from a common primitive ancestor. It is likely, however, that during evolution the actin sequences in these protozoa are not as well conserved as in other eukaryotic lineages.

Actins↗