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Biomedical subjects

H E Smith

Publications and source records attributed to H E Smith.

At least 19 recordsLinked to original sources
PubMed2001

The major bovine mastitis pathogens have different cell tropisms in cultures of bovine mammary gland cells.

We previously showed that Staphylococcus aureus cells adhered mainly to an elongated cell type, present in cultures of bovine mammary gland cells. Moreover, we showed that this adhesion was mediated by binding to fibronectin. The same in vitro model was used here, to study adhesion of other important mastitis pathogens. Like the S. aureus strains, the Streptococcus dysgalactiae strains adhered mainly to elongated cells, which seemed to be mediated by fibronectin binding. In contrast, Streptococcus uberis strains adhered mainly to cubic cells. Since the cubic cells did not express fibronectin and S. uberis cells bound fibronectin less efficiently, the adhesion of S. uberis cells was independent of fibronectin binding. Streptococcus agalactiae strains adhered poorly to both cell types. The specificity and efficiency of adhesion of Escherichia coli strains was strongly strain dependent. None of the S. agalactiae and E. coli strains tested was able to bind fibronectin efficiently. The results suggest that the different mastitis pathogens have different target cell specificities and use different mechanisms to adhere to cells of the bovine mammary gland.

Animals↗
PubMed2001

Protection of pigs against challenge with virulent Streptococcus suis serotype 2 strains by a muramidase-released protein and extracellular factor vaccine.

The efficacy of a muramidase-released protein (MRP) and extracellular factor (EF) vaccine in preventing infection and disease in pigs challenged either with a homologous or a heterologous Streptococcus suis serotype 2 strain (MRP+EF+) was compared with the efficacy of a vaccine containing formalin-killed bacterin of S. suis serotype 2 (MRP+EF+). The enhancement of the immune response by different adjuvants (a water-in-oil emulsion [WO] and an aluminium hydroxide-based adjuvant [AH]) and their side effects were also studied. The MRP and EF were purified by affinity chromatography. Pigs were vaccinated twice at three weeks and six weeks of age and challenged intravenously with virulent S. suis serotype 2 strains (MRP+EF+) at eight weeks of age. At challenge, the pigs vaccinated with MRP+EF/WO had high anti-MRP and anti-EF titres and were protected as effectively as pigs vaccinated with WO-formulated vaccines with bacterin. Eight of the nine pigs survived the challenge and almost no clinical signs of disease were observed. The titres obtained with the MRP+EF/AH vaccine were low and only two of the five pigs were protected. Pigs vaccinated with either MRP or EF were less well protected; three of the four pigs died after challenge but the clinical signs of disease were significantly less severe than those observed in the placebo-vaccinated pigs. The protective capacity of the bacterin/AH vaccine was very low, and the mortality among these pigs was as high as in the placebo-vaccinated pigs (80 per cent). Postmortem histological examination revealed meningitis, polyserositis and arthritis in the clinically affected pigs. The results demonstrate that a subunit vaccine containing both MRP and EF, formulated with the WO adjuvant, protected pigs against challenge with virulent S. suis type 2 strains.

Adjuvants, Immunologic↗
PubMed2001

Spatial variation in cartilage T2 of the knee.

Technical limitations imposed by resolution and B1 homogeneity have thus far limited quantitative in vivo T2 mapping of cartilage to the patella. The purpose of this study is to develop T2 mapping of the femoral/tibial joint and assess regional variability of cartilage T2 in the knee. Quantitative in vivo T2 mapping of the knee was performed on 15 asymptomatic adults (age, 22-44) using a 3T MR scanner. There is a consistent pattern of spatial variation in cartilage T2 with longer values near the articular surface. The greatest variation occurs in the patella, where T2 increases from 45.3 +/- 2.5 msec at a normalized distance of 0.33-67 +/- 5.5 msec at a distance of 1.0. These results demonstrate feasibility of performing in vivo T2 mapping of femoral tibial cartilage. Except for the superficial 15% where T2 values are lower, the spatial variation in T2 of femoral and tibial cartilage is similar to patellar cartilage.

Adult↗
PubMed2001

Sublingual immunotherapy.

Recent claims have been made that sublingual immunotherapy (SLIT) may be a viable alternative to injection immunotherapy (SIT). Animal studies show that when allergens are administered topically, they are handled differently, and IgE responses can be reduced. Most published studies of human SLIT have been small but show fairly consistent benefits on symptom scores, with few systemic side effects. Objective measures of allergen reactivity usually do not change. Relatively few subjects have been treated in SLIT trials compared with the numbers that would be required to validate new drug therapies. On the plus side, SLIT appears to work in adults and in children; it offers some logistic advantages and seems to be safe. Giving allergen by mouth rather than by injection should decrease the costs of immunotherapy, but the cumulative dose of allergen used in SLIT has been between 20 to 375 times the dose given in conventional SIT. Further cost-benefit analysis is needed. On the other hand, standard SIT is effective and is supported by better clinical and experimental evidence. The balance sheet for SLIT is improving, but on the current evidence, SLIT requires further evaluation before it could be recommended for use in routine clinical practice.

Administration, Sublingual↗
PubMed2001

Selection of virulence-associated determinants of Streptococcus suis serotype 2 by in vivo complementation.

Within Streptococcus suis serotype 2, pathogenic, weakly pathogenic, and nonpathogenic strains can be found. We introduced a genomic library of a pathogenic strain into a weakly pathogenic strain. After infection of the library into young piglets pathogenic transformants were selected. One specific transformant containing a 3-kb fragment of the pathogenic strain appeared to be dominantly enriched in diseased pigs. The observed enrichment was not tissue specific. The selected fragment, when introduced into two different weakly pathogenic strains, increased the virulence of these strains considerably. In contrast, introduction of the corresponding fragment of a weakly pathogenic strain had only minor effects on virulence. Nucleotide sequence analysis of the selected fragment of the pathogenic strain revealed the presence of two potential open reading frames, both of which were found to be mutated in the corresponding fragment of the weakly pathogenic strain. These data strongly suggest that the selected fragment contains determinants important for virulence.

Amino Acid Sequence↗
PubMed2001

Relatedness of Streptococcus suis isolates of various serotypes and clinical backgrounds as evaluated by macrorestriction analysis and expression of potential virulence traits.

We evaluated the genetic diversity of Streptococcus suis isolates of different serotypes by macrorestriction analysis and elucidated possible relationships between the genetic background, expression of potential virulence traits, and source of isolation. Virulence traits included expression of serotype-specific polysaccharides, muramidase-released protein (MRP), extracellular protein factor (EF), hemolysin activity, and adherence to epithelial cells. Macrorestriction analysis of streptococcal DNA digested with restriction enzymes SmaI and ApaI allowed differentiation of single isolates that could be assigned to four major clusters, named A1, A2, B1, and B2. Comparison of the genotypic and phenotypic features of the isolates with their source of isolation showed that (i) the S. suis population examined, which originated mainly from German pigs, exhibited a genetic diversity and phenotypic patterns comparable to those found for isolates from other European countries; (ii) certain phenotypic features, such as the presence of capsular antigens of serotypes 2, 1, and 9, expression of MRP and EF, and hemolysin activity (and in particular, combinations of these features), were strongly associated with the clinical background of meningitis and septicemia; and (iii) isolates from pigs with meningitis and septicemia showed a significantly higher degree of genetic homogeneity compared to that for isolates from pigs with pneumonia and healthy pigs. Since the former isolates are considered highly virulent, this supports the theory of a clonal relationship among highly virulent strains.

Animals↗
PubMed2000

Distribution of capsular types and production of muramidase-released protein (MRP) and extracellular factor (EF) of Streptococcus suis strains isolated from diseased pigs in seven European countries.

Streptococcus suis strains (n=411), isolated from diseased pigs in seven European countries were serotyped using specific antisera against serotype 1 to 28, and were phenotyped on the basis of their muramidase-released-protein (MRP) and extracellular-factor protein (EF) production. Overall, S. suis serotype 2 appeared to be most prevalent (32%), followed by serotype 9 (20%) and serotype 1 (12%). Serotype 2 was most frequently isolated in France, Italy and Spain, whereas serotype 9 was most frequently isolated in Belgium, The Netherlands and Germany. In the United Kingdom serotypes 1 and 14 were most frequently isolated. High percentages of S. suis serotype 1, 2, 1/2 and 14 strains, isolated from tissues associated with S. suis infections such as brain, serosa, joint, heart and organs expressed the EF-protein, indicating that in these serotypes expression of EF is likely to be associated with virulence. In contrast, strains belonging to serotype 7 and 9, isolated from tissues associated with S. suis infections did not produce EF. These results strongly suggest that in the serotypes 7 and 9 EF expression is not related to virulence. More than 80% of the S. suis serotype 9 strains produced an MRP* protein, a high molecular variant of the 136kDa MRP. Expression of MRP* in serotype 9 strains is possibly associated with virulence.

Age Factors↗
PubMed2000

The cps locus of Streptococcus suis serotype 2: genetic determinant for the synthesis of sialic acid.

The capsule of S. suis serotype 2 is composed of glucose, galatose, N-acetylglucosamine, rhamnose and sialic acid. Recently, we described a major part of the cps2 locus of S. suis serotype 2. Based on sequence homology genes encoding potential glucosyl-, galactosyl-, N-acetylglucosaminyl- and rhamnosyltransferase activities could be identified. However, we did not find genes involved in the synthesis of sialic acid. Here, we describe the cloning and characterization of a remaining part of the cps2 locus. Based on the establish sequence 11 potential genes, designated orf2L, orf2M, orf2N, cps2O to cps2T, orf2U and orf2V were identified. A gene homologous to genes involved in the polymerization of the repeating oligosaccharide unit (cps2O) as well as genes involved in the synthesis of sialic acid (cps2P to cps2T) were identified. Moreover, hybridizing experiments showed that the genes involved in the sialic acid synthesis are present in S. suis serotype 1, 2, 14, 27 and 1/2. The orf2M and orf2N regions showed similarity to proteins involved in the polysaccharide biosynthesis of other Gram-positive bacteria. However, these regions seemed to be truncated or were non-functional as the result of frame-shift or point mutations. At its 3;-end the cps2 locus contained two insertional elements (orf2U and orf2V), both of which seemed to be non-functional.

Bacterial Capsules↗
PubMed2000

A global profile of germline gene expression in C. elegans.

We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.

Animals↗
PubMed2000

Selection of recombinant antibodies specific for pathogenic Streptococcus suis by subtractive phage display.

A semisynthetic antibody phage display library was used to select recombinant antibodies directed against surface components of a pathogenic strain of Streptococcus suis serotype 2 and against extracellular factor (EF), a protein known to be exclusively associated with pathogenic S. suis serotype 2 strains. Three distinct monoclonal phage antibodies directed against conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage antibodies were isolated that recognized EF. To isolate antibody fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this procedure, only one distinct phage antibody was found, and it was shown to be directed against EF. This demonstrates the selectivity of the applied procedure and confirms that EF is indeed differentially expressed by pathogenic and nonpathogenic strains. It also shows that EF is a very dominant antigen in phage antibody selections.

Amino Acid Sequence↗
PubMed1999

The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells.

We recently described adhesion to and invasion of bovine mammary gland cells by Staphylococcus aureus in vitro. Here, we show that the levels of adhesion and invasion are dependent on the bacterial growth phase and are controlled by the agr locus. Incubation of exponential growth phase cells of S. aureus with mammary gland cells resulted in bacterial cell clumping. Strains of S. aureus deficient in expression of the fibronectin binding proteins (FnBPA and FnBPB) failed to clump and their ability to adhere to and to invade the bovine mammary gland cells is strongly reduced. This indicates that the fibronectin binding proteins are essential for S. aureus adhesion to and invasion of bovine mammary gland cells.

Adhesins, Bacterial↗
PubMed1999

Rapid PCR test for Streptococcus suis serotype 7.

Recent epidemiological studies on Streptococcus suis infections in pigs indicated that, besides serotypes 1, 2 and 9, serotype 7 is also frequently associated with diseased animals. For the latter serotype, however, no rapid and sensitive diagnostic methods are available. This hampers prevention and control programs. Here, we describe the development of a type-specific PCR test for the rapid and sensitive detection of S. suis serotype 7. The test is based on DNA sequences of capsular (cps) genes specific for serotype 7. These sequences were identified by cross-hybridization of several individual cps genes with the chromosomal DNAs of 35 different S. suis serotypes.

Animals↗
PubMed1999

Cell tropism of Staphylococcus aureus in bovine mammary gland cell cultures.

Staphylococcus aureus is one of the most important pathogens of the bovine mammary gland. The interaction of S. aureus with cells of the bovine mammary gland is considered to play an essential role in the pathogenesis. In this study, we identified a new target cell for S. aureus adhesion and invasion. For that purpose, cells which compose the alveoli of the mammary gland were cultured. In these cultures, two morphologically different cell types, elongated and cubic cells, were observed. Adhesion and invasion of S. aureus was studied using microscopical and microbiological methods. S. aureus adhered specifically and in large numbers (about 300 bacteria/cell) to the elongated cell type. No adhesion to the cubic cell type was observed. In addition, bacteria were also found intracellularly in the elongated cells, and enclosed in membrane vesicles. Adhesion and invasion were time dependent and reached maximum levels after 4 h. Invasion was strongly reduced by staurosporine and genistein. The newly identified target cell was further characterized.

Animals↗
PubMed1999

Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.

Animals↗
PubMed1999

Evaluation of bottlenecks in the late stages of protein secretion in Bacillus subtilis.

Despite a high capacity for secretion of homologous proteins, the secretion of heterologous proteins by Bacillus subtilis is frequently inefficient. In the present studies, we have investigated and compared bottlenecks in the secretion of four heterologous proteins: Bacillus lichenifomis alpha-amylase (AmyL), Escherichia coli TEM beta-lactamase (Bla), human pancreatic alpha-amylase (HPA), and a lysozyme-specific single-chain antibody. The same expression and secretion signals were used for all four of these proteins. Notably, all identified bottlenecks relate to late stages in secretion, following translocation of the preproteins across the cytoplasmic membrane. These bottlenecks include processing by signal peptidase, passage through the cell wall, and degradation in the wall and growth medium. Strikingly, all translocated HPA was misfolded, its stability depending on the formation of disulfide bonds. This suggests that the disulfide bond oxidoreductases of B. subtilis cannot form the disulfide bonds in HPA correctly. As the secretion bottlenecks differed for each heterologous protein tested, it is anticipated that the efficient secretion of particular groups of heterologous proteins with the same secretion bottlenecks will require the engineering of specifically optimized host strains.

Amino Acid Sequence↗
PubMed1999

Identification and characterization of the cps locus of Streptococcus suis serotype 2: the capsule protects against phagocytosis and is an important virulence factor.

To study the role of the capsule of Streptococcus suis serotype 2 in virulence, we generated two isogenic mutants disturbed in capsule production. For that purpose, we first cloned and characterized a major part of the capsular polysaccharide biosynthesis (cps) locus of S. suis serotype 2. Based on the established sequence, 14 open reading frames (ORFs), designated Orf2Z, Orf2Y, Orf2X, and Cps2A to Cps2K, were identified. Twelve ORFs belonged to a single transcriptional unit. The gene products of 11 of these ORFs showed similarity to proteins involved in polysaccharide biosynthesis of other gram-positive microorganisms. Nonencapsulated isogenic mutants were generated in the cps2B and cps2EF genes by insertional mutagenesis. In contrast to the wild-type S. suis serotype 2 strain, the nonencapsulated strains were highly sensitive to ingestion by porcine alveolar lung macrophages in vitro. More importantly, the nonencapsulated mutant strains were completely avirulent in young germfree pigs after intranasal inoculation. These observations indicate that the capsule of S. suis serotype 2 plays an essential role in the pathogenesis of S. suis serotype 2 infections.

Animals↗
PubMed1999

The cps genes of Streptococcus suis serotypes 1, 2, and 9: development of rapid serotype-specific PCR assays.

We developed three type-specific PCR assays for the rapid and sensitive detection of Streptococcus suis serotype 1 (plus 14), serotype 2 (plus 1/2), and serotype 9 strains in tonsillar specimens from pigs. The PCR primers were based on the sequences of type-specific capsular genes of S. suis serotype 1, 2, and 9 strains. We recently characterized a major part of the capsular biosynthesis (cps) locus of S. suis serotype 2. Here we extended these studies and characterized major parts of the cps loci of S. suis serotypes 1 and 9. Type-specific genes were identified by cross-hybridization experiments between the individual cps genes and chromosomal DNAs from the 35 different serotypes. Four genes of S. suis serotype 1 specifically hybridized with serotype 1 and 14 strains only. Five genes of S. suis serotype 2 specifically hybridized with serotype 2 and 1/2 strains only, and two genes of S. suis serotype 9 specifically hybridized with serotype 9 strains. Until now rapid and sensitive diagnostic tests were available only for pathogenic strains of serotype 2 and highly pathogenic strains of serotype 1. The serotype-specific PCR assays can therefore be useful tools for the identification of serotype 1, 14, 2, 1/2, and 9 strains both for diagnostic purposes and in epidemiological and transmission studies. Therefore, these tests may facilitate control and eradication programs.

Base Sequence↗