Phenolic-rich extracts from acerola, cashew apple and mango by-products cause diverse inhibitory effects and cell damages on enterotoxigenic Escherichia coli.

This study aimed to evaluate the inhibitory effects of phenolic-rich extracts from acerola (Malpighia emarginata D.C., PEA), cashew apple (Anacardium occidentale L., PEC) and mango (Mangifera indica L., PEM) by-products on distinct enterotoxigenic Escherichia coli (ETEC) strains. The capability of PEA and PEC of impairing various physiological functions of ETEC strains was investigated with multiparametric flow cytometry. Procyanidin B2 , myricetin and p-coumaric acid were the major phenolic compounds in PEA, PEC and PEM, respectively. PEA and PEC had lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (MIC: 31·25 mg ml-1 ; MBC: 62·5 mg ml-1 ) on ETEC strains than PEM (MIC and MIC: >1000 mg ml-1 ). PEA and PEC (15·6, 31·2, 62·5 mg ml-1 ) caused viable count reductions (P < 0·05) on ETEC strains after 24 h of exposure, notably the ≥3 log reductions caused by 62·5 mg ml-1 . The 24 h exposure of ETEC strains to PEA and PEC (31·2, 62·5 mg ml-1 ) led to high sizes of cell subpopulations with concomitant impairments in cell membrane polarization and permeability, as well as in enzymatic, respiratory and efflux activities. PEA and PEC are effective in inhibiting ETEC through a multi-target action mode with disturbance in different physiological functions.

A review of the current evidence of fruit phenolic compounds as potential antimicrobials against pathogenic bacteria.

Fruits are among the main natural sources of phenolic compounds (PC). These compounds exert important antioxidant properties primarily associated with the presence of hydroxyl groups in their molecular structure. Additionally, the antibacterial effects of fruit phenolic-rich extracts or individual PC commonly found in fruits have been an emerging research focus in recent years. This review discusses by first time the available literature regarding the inhibitory effects of fruit PC on pathogenic bacteria, including not only their direct effects on bacterial growth and survival, but also their effects on virulence factors and antibiotic resistance, as well as the possible mechanism underlying these inhibitory properties. The results of the retrieved studies show overall that the antibacterial effects of fruit PC vary with the target bacteria, type of PC and length of exposure to these compounds. The type of solvent and procedures used for extraction and fruit cultivar also seem to influence the antibacterial effects of phenolic-rich fruit extracts. Fruit PC have shown wide-spectrum antibacterial properties besides being effective antibiotic resistance modifying agents in pathogenic bacteria and these effects have shown to be associated with interruption of efflux pump expression/function. Furthermore, fruit PC can cause down regulation of a variety of genes associated with virulence features in pathogenic bacteria. Results of available studies indicate the depolarization and alteration of membrane fluidity as mechanisms underlying the inhibition of pathogenic bacteria by fruit PC. These data reveal fruit PC have potential antimicrobial properties, which should be rationally exploited in solutions to control pathogenic bacteria.

Binding to sulfatide and enterotoxicity of various Escherichia coli STb mutants.

Binding of the 48 amino acid polypeptide of the mature heat-stable Escherichia coli enterotoxin b (STb) to the functional receptor sulfatide (SFT) constitutes the first step in inducing secretory diarrhoea in the intestinal lumen of animals. The NMR structure of this toxin dictated the choice of amino acids for site-directed mutagenesis to delineate the binding site of STb to SFT. Amino acids facing the solvent either in the loop or the hydrophobic alpha-helix were selected. Seventeen site-specific mutants of STb toxin were produced and purified by high-pressure liquid chromatography. Enterotoxicity of the 17 mutants was determined using a rat loop assay and binding was evaluated using a microtitre plate binding assay. Both hydrophobic and electrostatic interactions are important for STb attachment. When mutations (F37K, I41S and M42S) were introduced into the hydrophobic alpha-helix to lessen hydrophobicity, binding activity and enterotoxicity decreased by more than sixfold. The loop defined by C21 and C36 also made specific contributions. Mutants generated at basic residues (K22, K23 and R29) within this region exhibited both reduced binding activities and reduced toxic activities. For all STb mutants constructed and analysed, when binding to SFT was reduced, a reduction in toxicity equivalent or greater was noted, indicating that binding to SFT is a step that precedes the toxic effect observed for STb toxin. Significantly, when the negatively charged D30 was substituted for either alanine or valine, the binding to SFT was about twice that of native STb, whereas the enterotoxicity was reduced by half.

In vitro binding characteristics and affinity for sulfatide of Escherichia coli STb enterotoxin.

It has previously been demonstrated that sulfatide (3'-sulfogalactosyl-ceramide), present at the surface of epithelial cells of the small intestine of pigs, interacts with the thermostable enterotoxin b (STb) produced by ETEC, and that this molecule is implicated in the mechanism of action of the toxin. However, few things are known about the affinity and physical characteristics of the interaction between these two macromolecules. In this study, using a microtiter plate binding assay (MPBA), we showed that STb toxin has a strong specificity for sulfatide and that this binding is dose-dependent and saturable. A very weak binding occurred with galactosyl-ceramide whereas attachment to 3'-sulfolactosyl-ceramide corresponded to 76% of the binding to sulfatide. STb toxin was shown to possess a lectin-like property; a significative binding was observed when a terminal beta-galactose was present in the glycosphingolipids tested and an increased binding was observed in presence of a sulfate group in position 3 on the galactose. These findings suggest that a sulfated galactosyl residue seems to represent the epitope recognized by the toxin. The reaction between sulfatide and STb toxin is also time and temperature dependent and is not affected by pH. The interaction was not inhibited by free sugars, sulfated polymers, glycolipids or free ions, but was partly inhibited by high concentrations of charged sugars. STb-sulfatide binding process is a low affinity interaction, as demonstrated by the determined Kd of 2-6 +/- 1.5 microM.

Distribution and expression of the astA gene (EAST1 toxin) in Escherichia coli and Salmonella.

The distribution and expression of the astA gene (EAST1 toxin) among 358 strains of Enterobacteriaceae were studied. The gene was found in 32.6% and 11.9% of Escherichia coli and Salmonella strains, respectively. The majority of E. coli EAST1-positive strains were found among EHEC (88.0%), EAggEC (86.6%), and A-EPEC (58.3%). The gene was present in 16.6% of E. coli strains without known virulence genes. There was no significant variation among the different serotypes of E. coli tested regarding the presence of the gene. For EPEC, 13.7% of the tested strains were astA-positive. Among atypical EPEC (eae+, bfp-, EAF-) and (eae+, bfp+, EAF-) 46.2 and 72.7%, respectively, were positive. The majority of the A-EPEC (87%) and EaggEC (83%) strains expressed the EAST-1 toxin as judged from Ussing chamber experiments. Of 32 EIEC strains studied, 2 possessed and expressed the gene as determined in Ussing chamber experiments. Among the Salmonella strains studied, five strains isolated from food were positive for astA and one strain of S. agona showed biological activity in Ussing chamber experiments.

Oligomerization of Escherichia coli enterotoxin b through its C-terminal hydrophobic alpha-helix.

Using a chemical cross-linker and gel electrophoresis or a dot blot overlay assay, we studied protein-protein interaction of STb toxin, a 48-residue amphiphilic polypeptide causing intestinal disorders. For the first time, we report on the oligomerization property of STb. This enterotoxin forms hexamers and heptamers in a temperature-independent fashion in presence or absence of its receptor (sulfatide) anchored in a 50-nm liposome or as a free molecule. Full STb structure integrity is necessary for its oligomerization as this process is not observed under reducing conditions in the presence of beta-mercaptoethanol. STb treatment with tetramethylurea (TMU) and different detergents prevented oligomerization. Site-directed mutagenesis decreasing overall STb hydrophobicity in the hydrophobic alpha-helix resulted in the incapacity to form oligomers. Taken together, these data suggest that the C-terminal hydrophobic alpha-helix corresponds to the domain of STb-STb inter-binding where hydrophobic interaction is involved.

[Bacterial enterotoxin receptors]. / Les récepteurs des entérotoxines bactériennes.

Bacterial enterotoxin receptors. Enteric bacterial toxins display a great diversity in their structure, molecular weight and mechanism of action. The interaction of enterotoxins with the intestinal mucosa either leads to a direct effect on the cell membrane or an effect on signal transduction within eukaryotic cells. However, before a toxin can affect a cell, it must after its secretion by a microorganism, recognise and bind to a specific surface molecule, its receptor. Membrane receptors of bacterial enterotoxins have been identified as protein, glycoprotein or glycolipid in nature. The chemical nature of the molecules acting as receptors is crucial and during evolution they have been carefully selected. Some toxins, after their interaction with a receptor molecule, will transduce a signal across the cell membrane while remaining at the cell surface. Other toxins, after this initial binding step with a receptor, will be internalised. Others can form pores leading to leakage of cellular components and cell lysis. Receptors that have been identified often comprise a saccharidic chain that is directly involved in the recognition and binding of the toxin. Today, models explaining toxin-receptor interactions are more complex, including multistep events. This review summarises the knowledge of the interactions between bacterial toxins and membrane receptors present on intestinal mucosa.

Actinobacillus pleuropneumoniae surface polysaccharides: their role in diagnosis and immunogenicity.

Actinobacillus pleuropneumoniae is an important pig pathogen that is responsible for swine pleuropneumonia, a highly contagious respiratory infection. Knowledge of the importance, composition and structural determination of the major antigens involved in virulence provides crucial information that could lead to the development of a rationale for the production of specific serodiagnostic tools as well as vaccine development. Thus, efforts have been devoted to study mainly A. pleuropneumoniae virulence determinants with special emphasis on the Apx toxins (for A. pleuropneumoniae RTX toxins). In comparison, little attention has been given to the surface polysaccharides, which include capsular polysaccharides (CPS) and cell-wall lipopolysaccharides (LPS). Here, we review current knowledge on CPS and LPS of A. pleuropneumoniae used as diagnostic tools to monitor the infection and as immunogens for inclusion in vaccine preparations for animal protection.

Inhibition of adherence of Actinobacillus pleuropneumoniae to porcine respiratory tract cells by monoclonal antibodies directed against LPS and partial characterization of the LPS receptors.

Actinobacillus pleuropneumoniae is the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia. We have previously identified the lipopolysaccharides (LPS) as the major adhesin of A. pleuropneumoniae involved in adherence to porcine respiratory tract cells. In the present study, adherence of A. pleuropneumoniae to porcine tracheal frozen sections was inhibited by homologous monovalent Fab fragments produced from monoclonal antibodies 5.1 G8F10 and 102-G02 directed, respectively, against the A. pleuropneumoniae serotype 1 or serotype 2 O-antigens. These results confirm the important role played by LPS in adherence of A. pleuropneumoniae and suggest that these adhesins might represent good vaccine candidates. We also investigated the presence of A. pleuropneumoniae receptors in tracheal cell preparations from piglets of four different breeds. Using Far-Western binding assays, we identified proteins recognized by whole cells of A. pleuropneumoniae reference strains for serotype 1 and 2, and local isolates belonging to the same serotypes, and also recognized by extracted LPS from both reference strains. We confirmed the proteinaceous nature of these LPS-binding molecules by their staining with Coomassie brilliant blue, sensitivity to proteinase K digestion, resistance to sodium m-periodate oxidation, and their inability to stain with glycoprotein-specific reagents. Four low-molecular-mass bands (14-17 kDa) seemed to correspond to histones. We also identified proteins at Mr 38,500 that could represent putative receptors for A. pleuropneumoniae LPS in swine respiratory tract cells.

Evidence that Escherichia coli STb enterotoxin binds to lipidic components extracted from the pig jejunal mucosa.

Escherichia coli strains producing the heat-stable enterotoxin STb cause diarrhoea in pigs, but little is known on the receptor binding step initiating the diarrhoeal process. In the present study, pig jejunal mucosa extracts were tested for the presence of binding component(s) for STb. Jejunal epithelial cells and the mucus layer were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated material was transferred to a polyvinylidene difluoride (PVDF) membrane and overlayed with STb. The results indicated that a band migrating with the tracking dye was bound by STb. This band was not stained by Coomassie blue and was thus regarded as non proteinic but rather as a lipidic component. Thus, total lipid extracts were obtained from the epithelial cells and the mucus layer. Compared to SDS-PAGE on 12% gels, a better separation of the low molecular mass components contained in these extracts was obtained using high-density Phastgel. Most of the components were detected following silver staining but not using Coomassie blue. Interestingly, commercially available pure glycolipids could also be visualized, after separation, only following silver staining. In the total lipid extracts, a band migrating in the 2.5-6.5 kDa range was observed. Using a monoclonal antisulfatide antibody, this band was recognized indicating that sulfatide was, in effect, present in the extract. When pure sulfatide was run on the same gels, it showed the same electrophoretic mobility. In addition, a dose dependent binding of STb to sulfatide could be observed. Taken together, these data suggested that sulfatide present on the jejunal mucosa, could represent a natural target binding molecule for STb.

Binding of Actinobacillus pleuropneumoniae lipopolysaccharides to glycosphingolipids evaluated by thin-layer chromatography.

The binding profile of Actinobacillus pleuropneumoniae serotypes 1 and 2 to various glycosphingolipids was evaluated by using thin-layer chromatogram overlay. A. pleuropneumoniae whole cells recognized glucosylceramide (Glcbeta1Cer), galactosylceramide (Galbeta1Cer) with hydroxy and nonhydroxy fatty acids, sulfatide (SO(3)-3Galbeta1Cer), lactosylceramide (Galbeta1-4Glcbeta1Cer), gangliotriaosylceramide GgO3 (GalNAcbeta1-4Galbeta1-4Glcbeta1Cer), and gangliotetraosylceramide GgO4 (Galbeta1-3GalNAcbeta1-4Galbeta1-4Glcbeta1Cer) glycosphingolipids. We observed no binding to globoseries, globotriaosylceramide Gb3, globoside Gb4, or Forssman Gb5 glycosphingolipids or to gangliosides GM1, GM2, GM3, GD1a, GD1b, GD3, and GT1b. The A. pleuropneumoniae strains tested also failed to detect phosphatidylethanolamine or ceramide. Interestingly, extracted lipopolysaccharide (LPS) of serotype 1 and serotype 2 as well as detoxified LPS of serotype 1 showed binding patterns similar to that of whole bacterial cells. Binding to GlcCer, GalCer, sulfatide, and LacCer, but not to GgO3 and GgO4 glycosphingolipids, was inhibited after incubation of the bacteria with monoclonal antibodies against LPS O antigen. These findings indicate the involvement of LPS in recognition of three groups of glycosphingolipids: (i) GlcCer and LacCer, where glucose is probably an important saccharide sequence required for LPS binding; (ii) GalCer and sulfatide glycosphingolipids, where the sulfate group is part of the binding epitope of the isoreceptor; and (iii) GgO3 and GgO4, where GalNacbeta1-4Gal disaccharide represents the minimal common binding epitope. Taken together, our results indicate that A. pleuropneumoniae LPS recognize various saccharide sequences found in different glycosphingolipids, which probably represents a strong virulence attribute.

Cloning and characterization of the esp region from a dog attaching and effacing Escherichia coli strain 4221 and detection of EspB protein-binding to HEp-2 cells.

The espA, espB and espD genes from enteropathogenic Escherichia coli were previously shown to be essential for triggering the signal transduction in infected host cells. We have cloned and determined the nucleotide sequences of the espA, espB and espD homologues from an E. coli strain (4221) isolated from a dog which manifested the attaching and effacing lesions in the small intestine. This strain is designated as a dog enteropathogenic E. coli. When comparing predicted amino acid sequences to those of the corresponding proteins from enteropathogenic E. coli O127, enterohemorrhagic E. coli serotype O26, enterohemorrhagic E. coli O157 and rabbit enteropathogenic E. coli, the EspADEPEC protein showed the same level of similarity (75% identity) with EspA of enteropathogenic E. coli O127 and rabbit enteropathogenic E. coli. The EspBDEPEC protein showed the highest similarity with the EspB of enteropathogenic E. coli O127 (99% identity). The EspDDEPEC protein showed 88% identity with the EspDEPEC. We constructed and purified a maltose-binding fusion protein containing the product of the entire espBDEPEC gene of the dog enteropathogenic E. coli strain 4221. Purified maltose-binding protein-EspBDEPEC fusion protein was shown to bind efficiently to HEp-2 cells in a localized fashion as shown by immunofluorescence microscopy. In addition, when the dog enteropathogenic E. coli strain 4221 was grown in tissue culture medium (DMEM) supplemented with serum, a secreted 36-kDa protein was identified by immunoblot analysis using a polyclonal antiserum against the maltose-binding protein-EspBDEPEC fusion protein.

Isolation and characterization of mini-Tn10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1.

Lipopolysaccharide (LPS) has previously been identified as the major adhesin of Actinobacillus pleuropneumoniae involved in adherence to porcine respiratory tract cells. The purpose of the present study was to isolate and characterize mutants in LPS biosynthesis by using a mini-Tn10 transposon mutagenesis system. Seven mutants appeared to possess a rough LPS (among which two had similar Southern blot profiles) while one mutant (#5.1) expressed the high-molecular-mass LPS, but as visualized by Tricine SDS-PAGE, showed an additional band in the core-lipid A region. The LPS mutants showed sensitivity to pig serum to various degrees, while the parent strain was serum-resistant. Use of piglet frozen tracheal sections indicated that, surprisingly, the rough LPS mutants adhered similarly or in greater numbers than the parent strain. However, the LPS mutant #5.1 adhered significantly less than the parent strain and was also less virulent in pigs. The gene affected by mini-Tn10 in LPS mutant #5.1 is galU, the structural gene for UTP-alpha-D-glucose-1-phosphate uridylyltransferase, involved in LPS core biosynthesis. Complementation analysis confirmed that the phenotypic characteristics of LPS mutant #5.1 are the result of the inactivation of the galU gene. Our data suggest that although the presence of O-antigen does not seem to be essential, an intact core-lipid A region might be required for adherence of A. pleuropneumoniae to porcine respiratory tract cells. To the best of our knowledge, these mutants represent the first isogenic mutants of A. pleuropneumoniae defective in LPS biosynthetic genes.

Evaluation of the protective efficacy of Actinobacillus pleuropneumoniae serotype 1 detoxified lipopolysaccharides or O-polysaccharide-protein conjugate in pigs.

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as the lipopolysaccharide (LPS). Experiments in our laboratory have shown that mice immunised with different A pleuropneumoniae serotype 1 LPS preparations were protected against a challenge with a virulent A pleuropneumoniae serotype 1 isolate. The purpose of the present study was to evaluate the protection of pigs against experimental A pleuropneumoniae infection following immunisation with two of these LPS preparations. Groups of five specific pathogen free (SPF) pigs were injected twice with one of the following antigen preparations: detoxified LPS, O-polysaccharide-BSA conjugate, a commercial bacterin, or PBS. Two weeks after the second injection, pigs were challenged intranasally with a virulent A pleuropneumoniae serotype 1 strain. Upon macroscopic examination, fibrino-haemorrhagic pleuropneumonia, compatible with A pleuropneumoniae infection, was observed in one to four pigs in each group. The more extensive lesions were present in control, unimmunised pigs and in animals vaccinated with the O-polysaccharide-BSA conjugate. The highest survival rate was recorded when the pigs had been immunised with detoxified LPS or the commercial bacterin. Taken together, our results suggest that a protection comparable with the one obtained with a commercial bacterin was observed when pigs were immunised with a single class of molecules, detoxified LPS. Most importantly, these results confirm the important role of A pleuropneumoniae LPS in protection against porcine pleuropneumonia. Finally, our results also support the idea that mice are not an appropriate model for the evaluation of porcine pleuropneumonia vaccines.

Sulfatide from the pig jejunum brush border epithelial cell surface is involved in binding of Escherichia coli enterotoxin b.

Using a quantitative dot blot overlay assay of polyvinylidene difluoride membranes, we investigated the ability of Escherichia coli heat-stable enterotoxin b (STb) to bind to various glycolipids of defined structure. STb bound strongly to acidic glycosphingolipids, including sulfatide (or 3'-sulfogalactosylceramide) and several gangliosides, but not significantly to their derivatives, galactosylceramide and asialogangliosides, respectively. STb exhibited the highest binding affinity for sulfatide. STb bound to pure sulfatide in a dose-dependent and saturable manner, with a detection level of a few nanograms. The binding was not inhibited by tetramethylurea, which is a strong disrupter of hydrophobic interactions, or by the anionic sulfated polymer of glucose, dextran sulfate, indicating that the binding is not due solely to either hydrophobic or ionic interactions via the sulfate group of the sulfatide. The specificity of the binding was confirmed by the finding that a 500-fold molar excess of sulfatide inhibited STb binding by approximately 45%, whereas no competition was obtained with galactosylceramide under the same conditions. Taken together, our data indicated that a galactose residue linked to a sulfate group is required for the binding specificity of STb. Then, total lipids extracted either from the mucous layer or from the epithelial cells of the pig jejunum brush border, the natural target of STb, were analyzed by thin-layer chromatography (TLC). Both extracts contained a lipidic molecule with a relative mobility on a TLC plate similar to that of the sulfatide standard. The migrated lipid extracted directly from a preparative TLC plate was confirmed to be sulfatide, as it was recognized by laminin, a sulfated glycolipid binding protein, and by a monoclonal antibody directed against sulfatide. In an overlay assay on PVDF membranes, STb bound to the sulfatide prepared from porcine jejunum as well as to the sulfatide standard. Thus, these findings suggest that the terminal oligosaccharide sequence Gal(3SO4)beta1- on sulfatide could mediate binding of STb to its target cells and, in support of a recent report (E. Rousset, J. Harel, and J. D. Dubreuil, Microb. Pathog. 24:277-288, 1998), probably terminal sialic acid residue on another glycosphingolipid. Moreover, pretreatment in the ligated intestinal loop assay with laminin or sulfatase altered the biological activity of STb. In summary, we present data indicating that sulfatide represents a functional receptor for the STb toxin.

Binding characteristics of Escherichia coli enterotoxin b (STb) to the pig jejunum and partial characterization of the molecule involved.

Escherichia coli heat-stable enterotoxin b (STb) causes severe diarrhoea in weaning piglets. STb most probably has to bind to intestinal epithelial cells in order to achieve its effect. Using biotinylated biologically active STb, we developed a semi-quantitative binding assay using indirect fluorescence microscopy. We demonstrated the attachment of the biotinylated toxin to microvilli of the pig jejunum. However, binding was abolished when biotinylated STb was either boiled or treated with 2-mercaptoethanol, treatments known to abolish biological activity. Different characteristics of STb attachment to the pig small intestine were determined. The reaction was rapid and reached maximum intensity after approximately 10 min. The binding was pH dependent showing an optimum at pH 5.8. Incubation at either 4 degrees C, 25 degrees C or 37 degrees C did not affect the binding. No competition was observed with non-biotinylated STb. However, preincubation of biotinylated STb with streptavidin conjugated to horseradish peroxidase completely abolished the binding. Pig tissues other than jejunum demonstrated binding towards STb including duodenum, ileum, caecum, colon, liver, lung, spleen and kidney. The molecule involved was then partially characterized. Metaperiodate treatment of the jejunum sections abrogated binding but protease treatment had no effect. Enzymatic treatments of jejunal sections demonstrated that N- and O-glycosidases, and several exoglycosidases did not affect binding, whereas reduced binding was observed with ceramide glycanase and alpha-glucosidase, and was completely abolished following neuraminidase treatment. Overall, our results suggest that in vitro STb binding was rapid, pH dependent, temperature independent, not restricted to jejunum and involves a molecule that seems to be composed of a ceramide moiety, terminal neuraminic acid and/or alpha-linked terminal glucose residue(s).

Immunochemical characterization of an IgG-binding protein of Streptococcus suis.

Several bacterial species express surface proteins with affinity for the constant region (Fc) of immunoglobulin (Ig) of different animal species. Previous studies from our group have reported the presence of an IgG-binding protein in various serotypes of Streptococcus suis. This molecule was also shown to bind in a non-immune fashion chicken IgY and to our knowledge this characteristic is unique. In the present study, by dot-blotting, we showed that the native protein, obtained by affinity chromatography, reacted more strongly with IgG from various animal species than the denatured material. Using a competitive enzyme-linked immunosorbent assay the affinity of the native 60-kDa protein (previously identified as a 52-kDa protein) towards IgG of various animal species was compared to pig IgG. Bovine, goat and human IgG were able to compete effectively with pig IgG whereas chicken IgY constituted a poor competitor. Peptide mapping analysis using denatured protein indicated that pig and bovine IgG recognized the same proteolytic fragment whereas chicken IgY did not. The smallest proteolytic fragment that retained the binding activity towards the IgG of the different animal species tested had a molecular mass of approximately 40 kDa. Fragments with Mr < 40 kDa showed specific binding activities. That is, the smallest fragment binding pig and bovine IgG had a Mr of 30 kDa whereas for goat and human IgG a fragment of less than 16 kDa still showed binding activity. Finally, we observed that antisera raised against a heat-shock protein of Pseudomonas aeruginosa reacted with the 60-kDa S. suis protein indicating that the S. suis 60-kDa protein is a member of the 60-kDa hsp family that possesses the characteristic of binding in a non-immune way mammalian IgG and chicken IgY.

Production of virulence-related proteins by Canadian strains of Streptococcus suis capsular type 2.

The production of muramidase-released protein (MRP), extracellular protein factor (EF) and hemolysin (suilysin) by 101 Canadian field strains of Streptococcus suis capsular type 2 is described. Most strains (72%) isolated from diseased pigs were MRP-EF- and only 1 strain was MRP+EF+. This strain was also the only 1 to produce the hemolysin. Thirteen strains (15%) were MRP+ EF- and only 3 strains were MRP* EF-. All the strains isolated from clinically healthy pigs as well as a bovine and 2 human isolates had a MRP-EF- phenotype. In addition, 7 strains (8%) had a MRPS phenotype, which had so far been described for S. suis capsular type 1. In conclusion, most Canadian field isolates of S. suis capsular type 2 tested in this study do not produce the virulence-related proteins described so far for this bacterial pathogen.

Isolation and characterization of LPS mutants of Actinobacillus pleuropneumoniae serotype 1.

The major adhesin of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, has been previously identified as lipopolysaccharide (LPS). The purpose of the present study was to isolate and characterize A. pleuropneumoniae LPS mutants. Screening of LPS mutants was performed with colony dot and sensitivity to novobiocin. One mutant obtained by colony dot (F19) and one mutant selected for its increased sensitivity to novobiocin (33.1) did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 O-antigen compared with the parent strain. Mutants F19 and 33.1 did not express high-molecular-mass LPS bands as determined in silver-stained SDS-PAGE gels. The core-lipid A region of mutant 33.1 and of the parent strain had similar relative mobilities and reacted with serum from a pig experimentally infected with the serotype 1 reference strain of A. pleuropneumoniae, while the same region in mutant F19 showed faster migration and did not react with this serum. Use of piglet tracheal frozen sections indicated that mutant F19 was able to adhere to piglet trachea as well as the parent strain, while mutant 33.1 adhered [half as much as] the parent strain. Finally, both LPS mutants were markedly less virulent in mice than the parent strain. Taken together, our observations support the idea that LPS is an important virulence factor of A. pleuropneumoniae.