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M A Smits

Publications and source records attributed to M A Smits.

At least 37 records · Page 2Linked to original sources
PubMed1997

Control of scrapie eventually possible?

After a brief description of the scrapie situation in the Netherlands, the technical progress made in aspects of scrapie diagnosis is reported. Emphasis is placed on the use of immuno-histochemistry (IHC) in the post-mortem histological diagnosis and on the recently published preclinical test for scrapie, in which IHC is applied to tonsillar biopsies. These two approaches use the same IHC technique and enable us to confirm suspected scrapie in individual animals, and for certain genotypes even in the preclinical phase. The tonsillar biopsy method could eventually lead to an infection- or PrPSc-detection method at flock level. Further work is required, including validation of the assay for various breeds, genotypes, and strains of the agent, and the conversion of the test into a more practical assay. The article continues with a discussion of several scrapie control strategies, describing briefly schemes tried in various countries, and elaborates on a proposed scrapie control scheme that could be suitable for the Netherlands. This scheme is essentially based on breeding for resistance, based on PrP genotyping.

Animals↗
PubMed1997

Endobronchial inoculation with Apx toxins of Actinobacillus pleuropneumoniae leads to pleuropneumonia in pigs.

To establish the role of the Apx toxins in the pathogenesis of porcine pleuropneumonia, specific-pathogen-free pigs were inoculated deeply endobronchially with either culture filtrates of Actinobacillus pleuropneumoniae serotype 8 or 9, culture filtrates depleted of the Apx toxins by affinity chromatography, depleted culture filtrate supplemented with purified recombinant Apx toxins (rApx), or purified rApx toxins alone. Results of these experiments indicate that ApxI, ApxIII, and, to a lesser extent, ApxII are the bacterial factors that trigger the development of clinical symptoms and lung lesions typical for porcine pleuropneumonia.

Actinobacillus pleuropneumoniae↗
PubMed1997

Virulent strains of Streptococcus suis serotype 2 and highly virulent strains of Streptococcus suis serotype 1 can be recognized by a unique ribotype profile.

The ribotype profiles of 42 different Streptococcus suis strains were studied. These strains belonged to five serotypes and differed in their virulence for pigs as well as in the expression of the muramidase-released protein and the extracellular protein factor. For the ribotyping, chromosomal DNAs were digested with EcoRI and were hybridized with a 1,066-bp ribosomal DNA probe. The hybridization patterns showed genetic heterogeneity within and between the serotypes. Pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1 could be recognized by their unique ribotype profiles. Nonpathogenic strains showed a high degree of genetic heterogeneity. Moreover, by comparing the 16S ribosomal DNA sequences of a number of S. suis strains, we were able to design two DNA probes which specifically hybridized with S. suis strains.

Bacterial Typing Techniques↗
PubMed1996

PrP genotype contributes to determining survival times of sheep with natural scrapie.

Several allelic variants of the sheep PrP gene are associated with scrapie susceptibility. However, it is not known whether, and to what extent, the PrP genotype contributes to determining survival times of scrapie sheep. We therefore determined the PrP genotype and life spans of over 50 Flemish and Swifter sheep within a single scrapie-affected flock. Eighty-three per cent of the scrapie sheep were homozygous for the PrP(VQ) allele (polymorphic amino acids at codons 136 and 171 are indicated) and these sheep died from scrapie at a mean age of 25 months. In sheep heterozygous for PrP(VQ), development of scrapie was delayed or did not occur. Sheep with at least one PrP(AR) allele, including PrP(VQ)/PrP(AR) sheep, did not develop scrapie. No scrapie sheep were found without a PrP(VQ) allele. We conclude that the PrP genotype contributes to determining survival times of sheep with natural scrapie. Additionally, we describe two novel sheep PrP allelic variants.

Animals↗
PubMed1996

Mutants of Streptococcus suis types 1 and 2 impaired in expression of muramidase-released protein and extracellular protein induce disease in newborn germfree pigs.

The contribution of muramidase-released protein (MRP) and extracellular factor (EF) to the virulence of Streptococcus suis type 1 and 2 infections was studied. For that aim, we constructed mutants of S. suis types 1 and 2 by inactivating the genes encoding MRP and EF. Moreover, we changed a type 2 strain producing the 110-kDa EF protein into a strain producing a modified protein (EF*) of increased molecular mass. The chromosomally located mrp and epf genes were inactivated by replacement recombination by using nonreplicative plasmids. Newborn germfree pigs were inoculated with pathogenic type 1 and 2 strains and with the isogenic mutant strains. Wild-type as well as mutant strains induced fever, specific signs of disease, and lesions. Moreover, all mutant strains could be reisolated from the central nervous system of infected pigs. These results showed that inactivation or alteration of the mrp and epj genes had no measurable effect on the pathogenicity of S. suis types 1 and 2.

Animals↗
PubMed1996

Influence of Actinobacillus pleuropneumoniae and its metabolites on porcine alveolar epithelial cells.

The effect of Actinobacillus pleuropneumoniae and its metabolites on the viability of porcine alveolar epithelial cells was studied by using a neutral-red uptake test. Alveolar epithelial cells were obtained from 5-week-old colostrum-deprived pigs. The purity of these cells as assessed by the modified Papanicolaou stain was 90 to 95%. Incubation of these cells with 10(6) CFU of a biotype 1 serotype 1 strain resulted in death of the alveolar epithelial cells within 1.5 h. A cytotoxic effect was also seen when alveolar epithelial cells were incubated with sterile culture supernatants of biotype 1 serotype 1, biotype 1 serotype 10, and biotype 2 serotype 2 strains or with ApxI, ApxII, or ApxIII produced by recombinant Escherichia coli. Incubation of alveolar epithelial cells with a knockout mutant of the biotype 1 serotype 1 parent strain which is unable to secrete Apx toxins or with its supernatant did not result in death of these cells. These results indicate that cytotoxicity is at least in part due to production of Apx toxins.

Actinobacillus pleuropneumoniae↗
PubMed1996

A specific and sensitive PCR assay suitable for large-scale detection of toxigenic Pasteurella multocida in nasal and tonsillar swabs specimens of pigs.

A polymerase chain reaction (PCR) assay for the detection of toxigenic Pasteurella multocida in nasal and tonsillar swab specimens collected from pigs was developed. Target DNA was isolated with guanidine thiocyanate and diatomite, and 2 primer sets derived from sequences in the gene that encodes the dermonecrotic toxin of P. multocida were used simultaneously. The method was adapted to microtiter plate format allowing large-scale use of the PCR assay. To identify false-negative test results caused by failure of amplification, a positive control template was constructed that was spiked to each DNA sample. The PCR assay was evaluated with clinical samples and compared with 2 routinely used methods for detection of toxigenic P. multocida: isolation from a selective agar and direct detection of the toxin in extracts of primary cultures by an enzyme-linked immunosorbent assay (ELISA). The sensitivity of the PCR assay was tested with 346 nasal and tonsillar swabs specimens collected from pigs of 9 herds known to be infected with toxigenic P. multocida. Toxigenic P. multocida was isolated from 22 specimens, only 28 specimens tested positive in ELISA, but 40 tested positive in the PCR assay; thus the PCR assay is the most sensitive of the 3 methods. The specificity of the PCR assay was tested with 372 swab specimens collected from pigs of 6 herds certificated to be free from toxigenic P. multocida. Toxigenic P. multocida was not isolated from any of these specimens, all tested negative in ELISA, and 370 tested negative in PCR. The 2 positive specimens came from 2 pigs of 1 litter and tested only weakly positive in the PCR assay. From these results, it was concluded that the PCR assay is not only highly sensitive but also highly specific.

Animals↗
PubMed1995

The CAMP effect of Actinobacillus pleuropneumoniae is caused by Apx toxins.

Actinobacillus pleuropneumoniae shows synergistic haemolysis when cocultured with Staphylococcus aureus on blood agar plates. This CAMP effect has been attributed to a discrete CAMP factor, but also to the A. pleuropneumoniae-RTX-toxins I, II, and III. We examined the CAMP effect of recombinant Escherichia coli strains that secreted each of these toxins, and of A. pleuropneumoniae mutant strains that were devoid of one or more these toxins. We found that the E. coli strains were CAMP positive, whereas the A. pleuropneumoniae strain devoid of functional toxin genes was CAMP negative. This demonstrated that the CAMP effect of A. pleuropneumoniae is caused by the toxins and that no CAMP factor per se exists.

Actinobacillus pleuropneumoniae↗
PubMed1995

Identification of five allelic variants of the sheep PrP gene and their association with natural scrapie.

Scrapie is a fatal neurodegenerative disease of sheep that belongs to the group of prion diseases found in humans and animals. The host encoded prion protein (PrP) plays a central role in the disease process. In the PrP genes of man, mice and sheep, polymorphisms have been found that are associated with disease susceptibility and pathogenesis. We have used denaturing gradient gel electrophoresis (DGGE) to detect polymorphisms in the sheep PrP gene. In addition to the already described polymorphisms at codons 136, 154 and 171, we identified a hitherto unknown G --> T transition at codon 171. This transition is responsible for a glutamine to histidine substitution. An arginine to glutamine substitution at this position has been described previously. DGGE allowed us to identify five different combinations of these polymorphisms within the PrP gene representing five allelic variants, which were cloned and sequenced. Based on the triplet sequences present at codons 136, 154 and 171 these allelic variants were designated PrPVRQ, PrPARR, PrPARQ, PrPARH and PrPAHQ. To determine the association of these allelic variants with natural scrapie, we screened 34 scrapie affected and 91 healthy control sheep of the Texel breed for the presence of these allelic variants. In these two groups, the five variants gave rise to 13 different genotypes. The distribution of the allelic variants among both groups showed marked differences. The PrPVRQ variant was present with high frequency in scrapie affected sheep, whereas the PrPARR variant was almost exclusively present in the healthy group. Two other variants, PrPARQ and PrPARH, were found in both groups with equal frequencies. The data obtained suggest modulation of disease susceptibility in these Texel sheep by at least five different PrP allelic variants, with the PrPVRQ and PrPARR alleles acting in a dominant, but opposite fashion over the PrPARQ and PrPARH alleles. The frequency of the PrPAHQ variant was too low to draw any conclusions.

Alleles↗
PubMed1995

High-efficiency transformation and gene inactivation in Streptococcus suis type 2.

An efficient electrotransformation system for Streptococcus suis type 2 is described. It is demonstrated that vectors based on the broad-host-range plasmid pWVO1 replicate in S. suis type 2. Transformation efficiencies of about 10(7) transformants per micrograms of plasmid DNA could be obtained. Derivatives of plasmid pBR322 containing S. suis chromosomal DNA did not replicate but integrated into the chromosome. Southern hybridization analysis revealed that double as well as single cross-over integration events had occurred. Double cross-over events occurred at a frequency of about 15%. With these transformation and integration systems, recombinant DNA technology can now be applied to this important pathogenic species.

Blotting, Southern↗
PubMed1995

Random amplified polymorphic DNA fingerprinting for rapid identification of leptospiras of serogroup Sejroe.

Eight leptospiral and three non-leptospiral primers were examined in the development of a random amplified polymorphic DNA (RAPD) fingerprinting method for identification of leptospiral serovars within the serogroup Sejroe. The strains examined comprised 19 reference strains of serogroup Sejroe, five field strains and 17 Leptospira borgpetersenii serovar hardjo type hardjobovis strains. Unique DNA banding patterns were obtained for each of the 17 reference strains with L. hardjo type hardjobovis-derived primers, and all clinical isolates were identified as L. hardjo type hardjobovis. Although the profiles were specific for each serovar, most serovars within serogroup Sejroe clustered into previously recognised genetic groups. RAPD fingerprinting is a simple, rapid method suitable for the identification of some Leptospira serovars.

Animals↗
PubMed1995

Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils.

The Actinobacillus pleuropneumoniae RTX-toxins ApxI, ApxII, and ApxIII are important virulence factors of this swine pathogen. It is hypothesized that the Apx toxins are deleterious to defense cells of the host, enabling the bacterium to infect the host. To confirm this, we studied the effect on porcine polymorphonuclear neutrophils of mutant strains of A. pleuropneumoniae that were devoid of Apx toxins. For this purpose, we developed a system for targeted mutagenesis of A. pleuropneumoniae based on the conditionally replicating plasmid pVE6063 and insertional mutagenesis by homologous recombination. Employing this system on the reference strain of serotype 1, a strain that secretes ApxI and ApxII, we generated mutant strains that were devoid of ApxI and/or ApxII. We compared the ability of the parent strain and the mutant strains to provoke an oxidative burst in porcine neutrophils and to kill these cells. The parent strain and mutants that secreted either ApxI or ApxII provoked an oxidative burst and killed the neutrophils, whereas mutant strains that were devoid of ApxI and ApxII did not. These experiments indicate the importance of ApxI and ApxII to these profound effects on neutrophils and emphasize the importance of ApxI and ApxII in pathogenesis.

Actinobacillus pleuropneumoniae↗
PubMed1995

Use of Multiscreen plates for the preparation of bacterial DNA suitable for PCR.

A rapid and inexpensive method for isolating bacterial DNA for use in PCR is described. The method is based on the guanidinium thiocyanate (GuSCN)-lysis method of Boom et al. (J. Clin. Microbiol. 28:495-503, 1990) and enables a multiple of 96 samples to be prepared in only one hour. We use Multiscreen plates and a vacuum manifold from Millipore. Clinical samples are lysed and washed in the wells of a Multiscreen plate, and DNA is eluted in a standard microplate. Purified DNA was recovered with high yields (over 25%). The method allows multichannel or robotic pipetting for both the sample preparation as well as for the PCR step. The method has been applied successfully to detect pathogenic Streptococcus suis type 2 in nasal and tonsil swab specimens of pigs.

Animals↗
PubMed1994

NAD-independent Actinobacillus pleuropneumoniae strains: production of RTX toxins and interactions with porcine phagocytes.

Actinobacillus pleuropneumoniae RTX toxin (Apx) production by A. pleuropneumoniae biotype 2 (NAD-independent) serotype 2 strains was studied. Western blot analysis of culture supernatants of all biotype 2 strains tested revealed the presence of a 103 kDa protein which reacted with a monoclonal antibody against ApxIIA. This protein was also recognized by sera of pigs infected with a biotype 2-serotype 2 strain. Furthermore, antibodies that could neutralize ApxIIA were present in these sera. Proteins corresponding to ApxIA or ApxIIIA were not detected. The effects of a biotype 1-serotype 2 and a biotype 2-serotype 2 strain and their metabolites on the oxidative activity of porcine pulmonary alveolar macrophages (PAM) and polymorphonuclear cells (PMN) were compared using a chemiluminescence (CL) technique. Viable bacteria of both biotypes stimulated the production of oxygen radicals by phagocytes. CL responses were higher for the biotype 1 than for the biotype 2 strain. After having reached a peak value, the oxidative activity decreased until a total inhibition was achieved. Inactivated washed bacteria had no influence on the oxidative activity of phagocytes. In contrast, heat labile factors in culture supernatants of both biotypes stimulated and inhibited the oxidative activity of PAM in a dose-dependent manner. Dilutions of supernatant up to 1/32 of the biotype 2 strain and up to 1/512 of the biotype 1 strain were toxic for PAM, while dilutions from 1/64 to 1/128 of the biotype 2 strain and from 1/1024 to 1/4096 of the biotype 1 strain stimulated the oxidative activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Actinobacillus pleuropneumoniae↗
PubMed1994

Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.

Actinobacillus pleuropneumoniae↗
PubMed1994

Production of Apx toxins by field strains of Actinobacillus pleuropneumoniae and Actinobacillus suis.

The three Apx toxins of Actinobacillus pleuropneumoniae have potential value for use in vaccines and diagnostic tests which will be species specific instead of serotype specific, provided that the Apx toxins are species specific and all field strains produce these toxins. We examined 114 A. pleuropneumoniae field strains and found that they secreted either ApxI, ApxII, ApxI and ApxII, or ApxII and ApxIII and secreted no other cytolytic activities. However, proteins similar to ApxI and ApxII were also produced by Actinobacillus suis.

Actinobacillus↗
PubMed1993

The gamma-tubulin gene of the malaria parasite Plasmodium falciparum.

By screening of cDNA and genomic libraries of Plasmodium falciparum with a DNA probe derived from the cognate beta-tubulin gene, gene Pf gamma tub has been identified that codes for gamma-tubulin, a newly discovered member of the tubulin superfamily that is indispensible for nuclear division and microtubule assembly [12]. Gene Pf gamma tub is not interrupted by introns and only present as a single-copy in the parasite genome. Its encoded amino acid sequence (452 amino acids; M(r) 50,560) has a 63% similarity to the gamma-tubulins encoded by Aspergillus nidulans, Schizosaccharomyces pombe, Drosophila melanogaster, Xenopus laevis and Homo sapiens. This figure is significantly (approx. 8%) lower than the average identity between the gamma-tubulins of the latter five species suggesting that during evolution the genes have been exposed to different selection pressures. The identity of gamma-tubulin to the Plasmodium falciparum encoded alpha- and beta-tubulins is 30 and 33%, respectively.

Amino Acid Sequence↗