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.

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.

Biochemical and serological characterization of Escherichia coli fimbrial antigen F165(2).

Mannose-resistant hemagglutinating fimbrial antigen F165 is produced by Escherichia coli strains associated with septicemia in piglets and calves. A fimbrial component with an M(r) of 17,200 as determined by SDS-PAGE was purified to homogeneity from F165-positive E. coli strain 4787 of serogroup O115. This fimbrial component of F165 antigen was named F165(2). Separation procedures included fast protein liquid chromatography with a Superose 12 column followed by ultracentrifugation and 0.15 M ethanolamine buffer (pH 10.5) dissociation. Upon removal of ethanolamine, the fimbrial component reassociated into fimbriae. Amino acid composition analysis indicated that the fimbrial component molecule comprised 158 amino acid residues of which 37.3% were hydrophobic. The amino acid composition and the isoelectric point (9.5) were readily distinguishable from those of F1 fimbriae. The amino acid sequence was determined for approximately 40% of the molecule. For the first 33 residues, the F165(2) sequence was identical to that of F1B fimbriae and very similar to that of F1C. Fimbriae F165(2) could nevertheless be differentiated antigenically from F1C fimbriae as demonstrated by the immunodot technique using cross-absorbed antisera.

Identification of a Streptococcus suis 60-kDa heat-shock protein using western blotting.

This study was initiated to investigate the presence of stress or heat shock proteins in Streptococcus suis. SDS-PAGE and Western blotting using polyclonal and monoclonal antibodies directed against different bacterial heat shock proteins demonstrated cross-reactivity with a protein with an apparent molecular mass of 60 kDa in all S. suis serotypes tested. The 60-kDa cross-reactive protein was present in virulent and avirulent strains of S. suis serotype 2 tested. A rabbit antiserum raised against the 60-kDa S. suis protein recognized the 60-65-kDa heat shock proteins in different Gram-positive and Gram-negative bacteria. Finally, the 60-kDa heat shock protein of S. suis was shown to be mostly secreted into the culture supernatant and, to a lesser extent, cell-associated. Growth under heat stress conditions (42 degrees C) increased the expression of the 60-kDa S. suis protein. This protein is, to our knowledge, the first common antigen found in different serotypes of S. suis.

Increase of capsular material thickness following in vivo growth of virulent Streptococcus suis serotype 2 strains.

Protein profile and capsular material thickness of Streptococcus suis serotype 2 strains were compared after in vitro and in vivo growth. Three virulent and one avirulent strains were used. These strains were grown in Brain Heart Infusion (BHI) broth, cells were collected by centrifugation, resuspended in a sterile saline solution and injected in diffusion chambers. The devices were then inserted in rat abdomens for 17 h. In vitro grown strains were also inoculated into fresh BHI broth and cultivated for 17 h at 37 degrees C. In vivo as well as in vitro grown bacteria were harvested by centrifugation, processed in a French pressure cell, treated with lysozyme and centrifuged to collect cell proteins for SDS-PAGE analysis. Transmission electron microscopy using polycationic ferritin labeling to stabilize capsular material was also carried out. No significant modification was noted in the protein profile for any strain after in vivo growth except for a 39 kDa protein of one virulent strain. On the other hand, an increase in thickness of capsular material was noted for the three in vivo grown virulent strains while no change was noted for the avirulent strain. This increase in capsular material thickness of virulent strains was accompanied by an increased resistance to killing by pig polymorphonuclear leukocytes. The capacity to produce more capsular material in vivo seems to be an attribute of some virulent S. suis serotype 2 strains.

Description of an albumin binding activity for Streptococcus suis serotype 2.

This study was undertaken to investigate the binding activity of Streptococcus suis serotype 2 to albumin. Using flow cytometry we observed a binding activity of S. suis to albumin for virulent as well as for avirulent isolates. Western immunoblots analysis revealed that a 39-kDa S. suis protein was responsible, at least in part, for this binding activity. This protein showed high N-terminal homology (95.6% for the first 23 residues) with a group A Streptococcus glyceraldehyde-3-phosphate dehydrogenase. Furthermore, the addition of albumin to the culture broth resulted in an increase in the virulence of S. suis strains in mice. These results suggest that an interaction with albumin could play a role in the pathogenesis of S. suis serotype 2 infections.

Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221.

We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221. When comparing the predicted amino acid sequence of the eaeDEPEC to that of the Eae proteins from enteropathogenic E. coli (EPEC), enterohaemorrhagic E. coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E. coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion. Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein. We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221. Binding of MBP-EaeDEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy. In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-EaeDEPEC fusion protein.

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.

A recombinant Escherichia coli heat-stable enterotoxin b (STb) fusion protein eliciting neutralizing antibodies.

STb is a heat-stable enterotoxin elaborated by enterotoxigenic Escherichia coli strains associated with weaning piglets and is responsible for diarrhoea in those animals. The maltose binding protein (MBP) of E. coli was used as a carrier for STb, a poorly immunogenic molecule. Constructions were produced where the gene coding for mature STb toxin (MBP-STb) and a fragment of the gene spanning the major epitopic region of STb (AA8-AA30) (MBP-STb2) were fused to malE gene coding for MBP. The fusion proteins accumulated in the periplasm and were detected with a polyclonal antibody raised against the purified toxin. MBP-STb induced secretion in the biological model whereas MBP-STb2 was non-toxic. Immunization of rabbits evoked an antibody response to STb for these two fusion proteins. However, only MBP-STb elicited antibodies that effectively neutralized the toxicity of pure STb toxin as determined in the rat loop assay.

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.

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.

Lipopolysaccharides of Actinobacillus pleuropneumoniae (serotype 1): a readily obtainable antigen for ELISA serodiagnosis of pig pleuropneumonia.

A saline extract of boiled-formalinized whole cells of Actinobacillus pleuropneumoniae serotype 1 reference strain (Shope 4074) has been previously used as the antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of swine pleuropneumonia. Phenol extraction of this crude extract permitted the recovery of LPS with long O-chains in the aqueous phase. This antigen was shown to be specific for serotypes 1, 9 and 11 as they all possess structurally similar O-chains. Immunoblotting was used to identify the fraction present in the crude extract of strain 4074 responsible for cross-reactions observed in ELISA with a serum raised against a serotype 3 strain of A. pleuropneumoniae. The specific reactions in ELISA were shown to be associated with long O-chain LPS and the cross-reactions to LPS with short O-chains. LPS seem to be an important antigen for A. pleuropneumoniae serotype 1 as all homologous sera tested reacted with it. This antigen is easily recovered from the crude extract and it can be used in minute amounts (1-6 micrograms) for ELISA serodiagnosis of pig pleuropneumonia.

Evaluation of long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 for the serodiagnosis of swine pleuropneumonia.

Long chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 5 have been evaluated and compared with a crude boiled extract (CBE) in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. The mean optical density (OD) obtained with the LC-LPS in ELISA using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotype 5 was not significantly different from that obtained with the CBE. However, sera from animals exposed to serotypes of A. pleuropneumoniae other than serotype 5 presented a significantly lower mean OD (P < 0.05) when the LC-LPS was used. As a consequence, it was demonstrated that a high percentage of non-specific cross-reactions were eliminated, without losing specificity. The specificity and the sensitivity of the LC-LPS- and CBE-ELISA were evaluated using two different cut-off values (the OD plus two and three standard deviations) (SD) obtained from 593 sera from negative herds. The LC-LPS appeared a more suitable antigen than the CBE, since the sensitivity and the specificity (obtained with both thresholds) were statistically improved (P < 0.01). A threshold of 0.244 (mean OD plus three SD) for the LC-LPS-ELISA seemed more suitable, since a sensitivity of 79% and a specificity of 97% was achieved. Nevertheless, it may be advisable to keep a buffer range (OD between 0.194 and 0.243) and to consider sera presenting values within this range as suspicious. In the present study, the complement fixation test presented a high specificity (97%) and a very low sensitivity (47%). A herd with animals presenting ELISA positive and CFT negative results in serology, along with the absence of suggestive lesions should not be considered as a non-infected herd.

Biochemical characterization of an antigenic saline extract of Actinobacillus pleuropneumoniae serotype 5 and identification of a serotype-specific antigen for ELISA serodiagnosis.

A saline extract of boiled-formalinized whole cells from a local strain (81-750; Quebec, Canada) of Actinobacillus pleuropneumoniae, serotype 5b was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of swine pleuropneumonia. Characterization of this crude extract was done and proteins, neutral sugars, hexosamines, and 2-keto-3-deoxyoctonate (KDO) were evaluated. On phenol extraction of the crude extract a serotype-specific antigen of polysaccharidic nature was recovered from the aqueous phase. This antigen was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue, silver and Schiff stainings. Immunoblots were done using sera of experimentally infected pigs that showed serotype specificity and cross-reactivity. Overall, the results indicate that the O-chain of lipopolysaccharides is a specific antigen that could be used in ELISA for the serodiagnosis of serotype 5 of A. pleuropneumoniae.

Evaluation of a saline boiled extract, capsular polysaccharides and long-chain lipopolysaccharides of Actinobacillus pleuropneumoniae serotype 1 as antigens for the serodiagnosis of swine pleuropneumonia.

A saline boiled extract (SBE), capsular polysaccharides (CPS) and long-chain lipopolysaccharides (LC-LPS) of Actinobacillus pleuropneumoniae serotype 1 have been evaluated in ELISA for the serodiagnosis of swine pleuropneumonia caused by this serotype. Mean optical densities (ODs) obtained with the three antigens using sera from negative herds as well as from animals experimentally and naturally exposed to A. pleuropneumoniae serotypes 1, 9 or 11 were not significantly different. The positive ELISA reaction with anti-serotypes 9 and 11 was unexpected with the CPS, which are supposed to be serotype-specific; LPS, and to a lesser extent proteins, were present in the CPS and appeared to be responsible for this reaction. In addition, sera from animals exposed to a field strain of A. pleuropneumoniae serotype 3 and to Actinobacillus suis presented a significantly lower mean OD (P < 0.001) when LC-LPS were used. Cross-reacting antigens consisted mainly of LPS core-lipid A present in the SBE and CPS. The specificity and the sensitivity of the ELISA were evaluated using three different cut-off values (the OD plus two, three and four times the standard deviation or SD) obtained with 667 negative sera. The diagnostic sensitivity was of 81% with the three antigens and the different thresholds. The diagnostic specificity was of 84, 86 and 88% for the mean plus two, three and four times the SD respectively using the SBE and the CPS, while that obtained with the LC-LPS was of 96, 98 and 99% using the same thresholds. In conclusion, LC-LPS make an easily obtainable antigen and seem to retain the best specificity while minimizing losses of sensitivity.

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.

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.

Immunization of mice against Streptococcus suis serotype 2 infections using a live avirulent strain.

In this study, the IgG response of mice injected with two virulent strains and one avirulent Streptococcus suis capsular type 2 strain was compared by Western blotting. The serum from mice immunized against the avirulent strain could recognize most proteins of the various strains tested and similar results were obtained with serum from mice injected with virulent strains. The live avirulent strain was injected twice (days 0 and 10) to groups of five mice, and four virulent strains from different geographical origins were used to challenge the animals. All mice, except one in one group, survived the challenge. These results suggest that a live avirulent strain could be used for immunization of swine, the natural host.

Comparison of pig, rabbit and mouse IgG response to Streptococcus suis serotype 2 proteins and active immunization of mice against the infection.

The aim of this study was to compare the IgG response of different animal species to Streptococcus suis serotype 2 proteins and to evaluate the immunogenic potential of these proteins in the mouse experimental model of infection. The protein profiles of ten different S. suis capsular type 2 isolates were compared by Western blotting using antisera produced in mice, rabbits and pigs against the reference strain. Strains were grown overnight in Todd-Hewitt broth, harvested by centrifugation, processed in a French press cell and digested with lysozyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was then performed and proteins transferred to nitrocellulose. The rabbit antiserum recognized seventeen common immunoreactive proteins, of which, proteins of 33, 44, 96, 122 kDa were present in all strains. Two, 128 and 136 kDa proteins were recognized by swine serum in many strains. An additional protein of 30 kDa was recognized by the mouse antiserum. These seven proteins, originating from the reference strain, were excised directly from polyacrylamide gels, mixed with incomplete Freund's adjuvant and given to groups of five mice on days 0 and 10. Immunoglobulin G response to each protein was monitored on day 20 using Western blots. Mice were then experimentally infected on day 21. Results indicated that vaccination with proteins of 33, 44, 128 and 136 kDa resulted in an IgG response and protection against the challenge with the reference strain, but gave only a partial protection against another virulent S. suis serotype 2 strain.