Biomedical subjects

Marcelo Gottschalk

Publications and source records attributed to Marcelo Gottschalk.

36 records · Page 2Linked to original sources

PubMed2005

Dilemma of virulence of Streptococcus suis: Canadian isolate 89-1591 characterized as a virulent strain using a standardized experimental model in pigs.

Virulence of Streptococccus suis capsular type 2 strain 89-1591 has been controversial in literature. A standardized experimental model with specific-pathogen free piglets was used for a new evaluation of this strain. Twenty-nine piglets were allotted in 4 separated groups. Group 1 consisted of negative control animals which received broth medium. Groups 2, 3, and 4 were intravenously challenged with 2 mL of S. suis, strains 1330, 89-1591, and 166', respectively. The strain 1330 is a recognized avirulent Canadian strain. The strain 166' is a reference French virulent isolate. Pigs inoculated with strain 1330 did not present clinical signs of a S. suis infection. Contamination in organs and bacterial blood circulation were rare and lesions were almost non-existent. Infection of pigs with S. suis strain 89-1591 (group 3) and 166' (group 4) caused severe clinical problems, animals infected with S. suis 166' were the most affected. Pigs presented with clinical signs such as high body temperature, lameness, nervous symptoms, and even mortality. Lesions associated with S. suis were numerous for both strains, but more evident in animals of group 4. It can be concluded that S. suis strain 89-1591 is virulent, although its virulence seems to be lower than that of the French strain. Results of an experimental infection with strain 89-1591 may depend on different factors such as the route of inoculation and the immunological status of the animals used. Using conventional animals, with an unknown status regarding previous S. suis infections, equivocal results may be obtained, and this may explain differences reported by some authors with the same strain.

Analysis of Variance↗

PubMed2004

Cloning and purification of the Streptococcus suis serotype 2 glyceraldehyde-3-phosphate dehydrogenase and its involvement as an adhesin.

Streptococcus suis serotype 2 is a swine pathogen responsible for diverse diseases and may be present in the tonsils of pigs which show no sign of illness. Because adhesion to host cells may be important in the carrier state, this study was undertaken to characterize a 39 kDa surface protein identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH), possibly implicated in the adhesion of the bacteria. The gene encoding for the GAPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 336 amino acid polypeptide exhibiting 95% sequence identity with the GAPDH from Streptococcus pyogenes and from other streptococci. Using the Qiaexpress expression plasmids, the gapdh gene was inducibly overexpressed in E. coli to produce GAPDH with a hexahistidyl N-terminus to permit its purification. The (His)6GAPDH protein was found to possess functional GAPDH enzymatic activity after the purification. An adherence assay with S. suis and porcine tracheal rings pre-incubated with (His)6GAPDH and non-incubated rings was showed a significant reduction in the adhesion of S. suis in the (His)6GAPDH pre-incubated rings compared to the non-incubated rings. The GAPDH protein of S. suis seems to be involved in the first steps of the bacterial adhesion to host cells.

Amino Acid Sequence↗

PubMed2004

Extracellular virulence factors of streptococci associated with animal diseases.

A virulence factor denotes a bacterial product or strategy that contributes to virulence or pathogenicity. Streptococci produce a variety of protein toxins and enzymes that are capable of killing host cells and breaking down cell constituents, presumably to provide nutrients for the bacteria or to promote their spread. Some of these secreted products are hemolysins, streptokinases, hyaluronidases, exotoxins and proteases. In some cases, they play an important role in resistance to the host immune system, acting alone or in combination with cell-associated virulence factors (such as the capsule and surface proteins). Thus, the virulence of streptococci is considered as a multifactorial process. In contrast to well known human pathogens, and in spite of their veterinary importance, knowledge of virulence factors of most animal disease-associated streptococci is limited or almost inexistent. In the present article, the available information regarding the extracellular virulence factors of the most important animal disease-related streptococci is reviewed.

Animals↗

PubMed2004

Acquisition of host plasmin activity by the Swine pathogen Streptococcus suis serotype 2.

In this study, the plasminogen-binding activity of Streptococcus suis serotype 2 was investigated. Bound human plasminogen was activated by purified streptokinase, urokinase, or Streptococcus dysgalactiae subsp. equisimilis culture supernatant. Both human and porcine plasminogen were bound by S. suis. Binding was inhibited by epsilon-aminocaproic acid, and the plasminogen receptor was heat and sodium dodecyl sulfate resistant. One of the receptors was identified as glyceraldehyde-3-phosphate dehydrogenase. S. suis-associated plasmin activity was capable of activating free plasminogen, which in turn could contribute to degradation of fibronectin. This is the first report on the plasminogen-binding activity of S. suis. Further studies may reveal a contribution of this activity to the virulence of S. suis.

Animals↗

PubMed2004

Invasion of porcine brain microvascular endothelial cells by Streptococcus suis serotype 2.

Streptococcus suis is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of S. suis infection has not been completely defined. However, in order to cause meningitis, S. suis has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of S. suis serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European S. suis serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of S. suis seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable S. suis could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by S. suis required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by S. suis.

Actin Cytoskeleton↗

PubMed2004

Encapsulated Streptococcus suis inhibits activation of signaling pathways involved in phagocytosis.

Streptococcus suis capsular type 2 is an important zoonotic agent of meningitis. Previous studies reported that, in contrast to nonencapsulated mutants, encapsulated S. suis is able to resist phagocytosis. However, the mechanisms by which S. suis avoids phagocytosis are unknown. To elucidate the signaling pathway(s) involved in S. suis antiphagocytosis, we compared the ability of an encapsulated strain and its nonencapsulated mutant to induce the activation of Akt and protein kinase C (PKC), which are downstream kinases of the phosphatidylinositol 3-kinase (PI-3K) pathway, known to be involved in the phagocytosis processes. The results demonstrated high levels of Akt and PKCalpha phosphorylation after infection of J774 macrophages with the nonencapsulated mutant, whereas the encapsulated strain showed reduced activation of PI-3K/Akt/PKCalpha signaling pathway, as well as several protein tyrosine events. These results correlated with the number of intracellular bacteria. Macrophages pretreated with specific PI-3K or PKC inhibitors showed reduced levels of Akt and PKCalpha phosphorylation, resulting in 50% reduction of phagocytosis. The role of phosphatases in the antiphagocytic mechanisms was evaluated by using phosphatase inhibitors, as well as SHP-1-deficient macrophages. Only in the absence of SHP-1 did the phagocytosis of encapsulated S. suis significantly increase, leading to Akt phosphorylation levels similar to those observed with the nonencapsulated strain, indicating activation of this important SH2 domain-containing tyrosine phosphatase by encapsulated S. suis. Finally, when purified S. suis capsular polysaccharide (CPS) was added to macrophages, no phosphorylation events were observed. In addition, CPS and encapsulated S. suis were able to inhibit the uptake of the nonencapsulated mutant. These results suggest the importance of CPS in the mechanisms, whereby S. suis downmodulates phagocytosis.

Animals↗

PubMed2004

Development and comparison of enzyme immunoassays for diagnosis of Chagas' disease using fixed forms of Trypanosoma cruzi (Epimastigotes, Amastigotes, and Trypomastigotes) and assessment of antigen stability for the three assays.

Three enzyme immunoassays (EIAs) for diagnosis of Chagas' disease were developed with fixed forms of Trypanosoma cruzi using a panel of 435 sera from the following groups: Venezuelan subjects positive by immunofluorescence (n = 70), Venezuelan healthy controls (n = 85), healthy Canadians (n = 166), and subjects with other parasitic diseases (n = 114). All assays achieved 100% sensitivity and reasonable specificity for amastigotes (97.6%), epimastigotes (98.3%), and trypomastigotes (99.3%). The fixed-trypomastigote assay was stable over 4 months at 4 degrees C and room temperature. These data suggest that a fixed-trypomastigote EIA may be a suitable candidate for blood bank screening.

Animals↗

PubMed2004

Serotype III Streptococcus agalactiae from bovine milk and human neonatal infections.

Streptococcus agalactiae (group B streptococcus [GBS]) causes invasive human infections and bovine mastitis. This study examined the genetic relationship between bovine and human serotype III GBS by using molecular techniques that classify human serotype III GBS into four distinct phylogenetic lineages. Bovine serotype III GBS were largely contained in two lineages, which are distinct from the two major lineages (restriction digest types III-2 and III-3) that infect human neonates. One of the bovine lineages closely resembles the human III-1 lineage, whose members occasionally cause human neonatal infections. The bovine strains in the other lineage characteristically have an initiation factor IF2 gene (infB) H allele and multilocus sequence types that are not found in human GBS strains. Evidence suggests that this "H allele" lineage is related to the human III-3 lineage. These results support the assertion that human and bovine GBS are largely unrelated and provide further insight into the genetic relation between human and bovine GBS.

Alleles↗

PubMed2003

Cloning and partial characterization of the gene encoding the putative elongation factor Ts of Streptococcus suis serotype 2.

Streptococcus suis infection has a substantial impact on the swine industry. In addition, S. suis serotype 2 is recognized as a zoonotic agent. In this paper, we report the cloning and complete sequence of the gene coding for the putative elongation factor Ts ( tsf-like) of S. suis. The putative tsf gene seems to be transcribed from a promoter located within the cloned DNA fragment, as its expression is not dependent on insertional orientation within the plasmid. One copy of the tsf gene was detected in the chromosome of S. suis by Southern blot analysis. Interestingly, the elongation factor Ts expressed by all reference strains of all S. suis serotypes were antigenically similar, as determined by Western blot.

Antibodies, Bacterial↗

PubMed2003

Pro-inflammatory cytokine and chemokine release by human brain microvascular endothelial cells stimulated by Streptococcus suis serotype 2.

Streptococcus suis serotype 2 is a world-wide agent of diseases among pigs including meningitis, septicemia and arthritis. This microorganism is also recognized as an important zoonotic agent. The pathogenesis of the meningitis caused by S. suis is poorly understood. We have previously shown that S. suis is able to adhere to human brain microvascular endothelial cells (BMEC), but not to human umbilical vein endothelial cells (HUVEC). The objective of this work was to study the ability of S. suis serotype 2 to induce the release of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1); IL-6 and the chemokines IL-8 and monocyte chemotactic protein-1 (MCP-1) by human BMEC and HUVEC, using a sandwich enzyme-linked immunosorbent assay. S. suis was able to stimulate the production of IL-6, IL-8 and MCP-1 by BMEC but not HUVEC, in a time- and concentration-dependent manner. Bacterial cell wall components were largely responsible for such stimulation. The human and pig origin of strains does not seem to affect the intensity of the response; indeed, a very heterogeneous pattern of cytokine and chemokine production was observed for the different strains tested in this study. In situ production of cytokines and chemokines by BMEC may be the result of specific adhesion of S. suis to this cell type, with several consequences such as increased recruitment of leukocytes and an increase in the blood-brain barrier permeability.

Brain↗

PubMed2003

Evaluation and field validation of PCR tests for detection of Actinobacillus pleuropneumoniae in subclinically infected pigs.

Eight PCR tests were evaluated for their abilities to detect Actinobacillus pleuropneumoniae in swine tonsils. At first they were compared regarding their specificities by using A. pleuropneumoniae and related bacterial species and their analytical sensitivities by using tonsils experimentally infected in vitro. PCRs were carried out both directly with tonsil homogenates (direct PCR) and after culture of the sample (after-culture PCR). Most tests demonstrated good specificities; however, some tests gave false-positive results with some non-A. pleuropneumoniae species. High degrees of variation in the analytical sensitivities among the tests were observed for the direct PCRs (10(9) to 10(2) CFU/g of tonsil), whereas those of most of the after-culture PCRs were similar (10(2) CFU/g of tonsil). In a second phase, the effects of sample storage time and storage conditions were evaluated by using tonsils from experimentally infected animals. Storage at -20 degrees C allowed the detection of the organism for at least 4 months. Finally, the omlA PCR test described by Savoye et al. (C. Savoye et al., Vet. Microbiol. 73:337-347, 2000) and the commercially available Adiavet App PCR test were further validated with field samples. Their effectiveness was compared to those of standard and immunomagnetic separation-based methods of bacterial isolation. In addition, a comparison of tonsil biopsy specimens (from living animals) and whole tonsils (collected at the slaughterhouse) was also conducted. A. pleuropneumoniae was neither isolated nor detected by PCR from a herd serologically negative for A. pleuropneumoniae. PCR was more sensitive than the standard isolation method with whole tonsils from three infected herds. After-culture PCR offered the highest degree of sensitivity (93 and 83% for the omlA and Adiavet App PCRs, respectively). The PCR detection rate was higher with whole tonsils than with tonsil biopsy specimens. Good agreement (kappa = 0.65) was found between the presence of A. pleuropneumoniae in tonsils and the individual serological status of the animals.

Actinobacillus Infections↗

PubMed2003

Serotypes of Streptococcus agalactiae cultured from dairy milk samples in Québec.

Streptococcus agalactiae remains an important pathogen of dairy herds in Québec, but data about antigenic characteristics of this microorganism are sparse. This study was conducted to determine the variety of S. agalactiae serotypes in dairy herds in Québec. Two hundred and ninety-five isolates cultured from the milk of individual cows from 7 regions of Québec were serotyped. Sixty-two percent of the isolates were untypable. Among the 38% of typeable isolates, serotype III was found most frequently. In conclusion, the heterogeneity found among antigenic determinants of isolates from bovine milk suggests that an immunological method for the detection of S. agalactiae performed directly on bovine milk would not be a practical approach.

Animals↗

PubMed2002

Streptococcus suis interactions with the murine macrophage cell line J774: adhesion and cytotoxicity.

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since one hypothesis of the pathogenesis of S. suis infection is that bacteria enter the bloodstream and invade the meninges and other tissues in close association with mononuclear phagocytes, the objective of the present study was to evaluate the capacity of S. suis type 2 to adhere to macrophages. An enzyme-linked immunosorbent assay technique was standardized to simply and accurately measure the rate of bacterial attachment to phagocytic cells. Results were confirmed by plate counting. Adhesion was dependent on bacterial concentration and incubation time and was not affected by cytochalasin pretreatment of macrophages. Inhibition studies showed that the sialic acid moiety of the S. suis capsule would be, at least in part, responsible for bacterial recognition by macrophages. Serum preopsonization of bacteria increased adhesion levels. Complement would be partially implicated in the serum-enhanced binding of S. suis to cells. Adhesion varied among different S. suis type 2 isolates. However, high bacterial concentrations of several isolates were cytotoxic for cells, and these cytotoxic effects correlated with suilysin production. Indeed, hemolytic strain supernatants, as well as purified suilysin, reproduced cytotoxic effects observed with live bacteria, and these effects were inhibited by cholesterol pretreatment. Bacterial adhesion and cytotoxicity were confirmed by scanning and transmission electron microscopy. We hypothesize that attachment of bacteria to phagocytes could play an important role in the pathogenesis of S. suis infection by allowing bacterial dissemination and causing a bacteremia and/or septicemia. This interaction could also be related to the activation of the host inflammatory response observed during meningitis.

Animals↗

PubMed2002

Genetic diversity of Streptococcus suis strains isolated from pigs and humans as revealed by pulsed-field gel electrophoresis.

The genetic diversity of 123 Streptococcus suis strains of capsular types 2, 1/2, 3, 7, and 9, isolated from pigs in France and from humans in different countries, was evaluated by pulsed-field gel electrophoresis (PFGE) of DNA restricted with SmaI. The method was highly discriminative (D = 0.98), results were reproducible, and the PFGE analysis was easy to interpret. Among all S. suis strains, 74 PFGE patterns were shown. At 60% homology, three groups (A, B, and C) were identified, and at 69% homology, eight subgroups (a to h) were observed. Strains isolated from diseased pigs or from humans were statistically clustered in group B, especially in subgroup d. By contrast, S. suis strains isolated from clinically healthy pigs were preferentially included in subgroup b of group A. Relationships could be established between capsular types 1/2, 3, and 9 and groups A, e, and B, respectively. S. suis strains isolated from humans were homogeneous, and a very high level of association between these strains and four DNA patterns was observed. The PFGE used in this study is a very useful tool for evaluating the genetic diversity of S. suis strains, and it would be used for epidemiological investigations.

Animals↗

PubMed2002

Genetic diversity of Streptococcus suis serotypes 2 and 1/2 isolates recovered from carrier pigs in closed herds.

The aim of this study was to compare, by randomly amplified polymorphic DNA (RAPD), the diversity of Streptococcus suis serotypes 1/2 and 2 isolates recovered at slaughter houses from the tonsils of clinically healthy pigs. The pigs belonged to herds with or without clinical signs of S. suis disease. Overall, a low diversity was observed among isolates of serotype 1/2. A representative isolate recovered from a diseased animal presented a relatively high similarity (85%), with most isolates recovered from carrier pigs, from herds either with or without clinical signs of S. suis disease. For serotype 2 isolates, a relatively high degree of heterogeneity was observed in the whole population. Two subpopulations were observed for serotype 2 isolates, which arose from herds with clinical signs. Interestingly, the representative isolate coming from the diseased pig was included in a small closed cluster, with 2 isolates recovered from carrier pigs belonging to the same herd. On the other hand, most of the S. suis serotype 2 isolates originating from herds with no history of S. suis disease, were closely related (90% similarity). Furthermore, they presented different RAPD patterns from those originating from animals from the herd presenting S. suis clinical signs due to this serotype. Results suggest that, in the herds studied, clinical manifestations due to serotype 2 are probably related to the virulence of a specific isolate. Conversely, for the herd affected with serotype 1/2, clinical manifestations of the disease were more likely to be the result of inherent herd factors than the virulence of the specific isolate.

Abattoirs↗

PubMed2000

Interactions between Streptococcus suis serotype 2 and different epithelial cell lines.

Streptococcus suis is an important swine pathogen responsible for cases of sudden death, septicaemia, meningitis, endocarditis and pneumonia. It is also recognized as a zoonotic agent in people occupationally exposed to pigs or pig products. Knowledge on virulence factors of S. suis serotype 2 is limited and the pathogenesis of the infection is poorly understood. It has been suggested that the disease due to S. suis serotype 2 begins with colonization of the nasopharyngeal epithelium, followed by either spread within the respiratory tract or invasion of the bloodstream. The mechanisms involved in the access of bacteria from the bloodstream to the central nervous system are unknown. It is possible that epithelial cells of the choroid plexus also play an important role in the pathogenesis of the meningitis. Different interactions (adhesion, invasion and toxic effects) of S. suis serotype 2 with epithelial cell lines [LLC-PK1, PK(15), A549, HeLa and MDCK] were studied and compared to those of a human pathogen which also causes meningitis, group B Streptococcus (GBS). The results showed that S. suis serotype 2, in contrast to GBS, is able to adhere to but not to invade epithelial cells. The adhesin(s) involved seem(s) to be partially masked by the capsule and are a part of the cell wall. The haemolysin produced by S. suis serotype 2 is responsible for a toxic effect observed on epithelial cells. The results described give additional evidence that pathogenesis of the infection differs between S. suis and GBS. In particular, it is possible that suilysin-positive S. suis strains use adherence and cell injury, as opposed to direct cellular invasion, as part of a complicated multistep process which leads to bacteraemia and meningitis in pigs.

Animals↗

PubMed1998

Streptococcus suis serotype 2 mutants deficient in capsular expression.

Streptococcus suis serotype 2 is responsible for a wide variety of porcine infections. In addition, it is considered a zoonotic agent. Knowledge about the virulence factors for this bacterium is limited but its polysaccharide capsule is thought to be one of the most important. Transposon mutagenesis with the self-conjugative transposon Tn916 was used to obtain acapsular mutants from the virulent S. suis type 2 reference strain S735. Clones were screened by colony-dot ELISA with a monoclonal antibody specific for a type 2 capsular epitope and clones that failed to react with the antibody were characterized. Two mutants, 2A and 79, having one and two Tn916 insertions respectively, were chosen for further characterization. Absence of capsule was confirmed by coagglutination, capillary precipitation and capsular reaction tests and by transmission electron microscopy. Absence of capsular polysaccharides correlated with increased hydrophobicity and phagocytosis by both murine macrophages and porcine monocytes compared to the wild-type strain. Furthermore, both mutants were shown to be avirulent in murine and pig models of infection. Finally, mutant 2A was readily eliminated from circulation in mice compared to the wild-type strain, which persisted more than 48 h in blood. Thus, isogenic mutants defective in capsule production demonstrate the importance of capsular polysaccharides as a virulence factor for S. suis type 2.

Agglutination Tests↗

PubMed1997

Characterization and protective activity of a monoclonal antibody against a capsular epitope shared by Streptococcus suis serotypes 1, 2 and 1/2.

A monoclonal antibody (mAb Z3) was produced using BALB/c mice immunized with whole cells of Streptococcus suis serotype 2 reference strain S735. Screening by dot-ELISA showed that mAb Z3, of isotype IgG2b, reacted only with reference strains and field isolates of S. suis serotypes 1, 2 and 1/2. The recognized epitope was demonstrated to be polysaccharide in nature by periodate oxidation, and located in the capsule, since mAb Z3 reacted with purified capsular material by immunoblotting and was able to stabilize the capsule as shown by electron microscopy. Further characterization indicated that mAb Z3 may react specifically with the sialic acid moiety of the capsule, a common constituent of the polysaccharidic capsular material of the three capsular types, since sialidase-treated cells did not react with mAb Z3 in immunoblotting or indirect ELISA. Purified mAb Z3 was shown to significantly increase the rate of phagocytosis of S. suis cells by porcine monocytes and to activate the clearance of bacteria from the circulation in experimentally infected mice. However, mAb Z3 only offered partial protection to mice challenged with a minimal lethal dose. Thus, even though the capsule of S. suis seems to be an important virulence factor, the epitope recognized by mAb Z3 does not appear to be involved in complete protection against infection.

Animals↗