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Marcelo Gottschalk

Publications and source records attributed to Marcelo Gottschalk.

At least 19 recordsLinked to original sources
PubMed2007

Detection and molecular typing of Streptococcus suis in tonsils from live pigs in France.

Streptococcus suis is an important pathogen of swine, causing meningitis, arthritis, polyserositis, septicemia, and sudden death in weaning piglets as well as fattening pigs. Recently, 3 molecular tests have been developed in our laboratory: a multiplex polymerase chain reaction (m-PCR) assay for the detection of S. suis species and serotypes 2 and 1/2, and 2 molecular typing methods, pulsed-field gel electrophoresis and an approach based on PCR amplification of a fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S rDNA intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis (ISR-RFLP). In the present study, we used these tests to analyze tonsil samples from clinically healthy pigs and to identify individual isolates of S. suis during epidemiologic investigations of 8 related herds with a history of septicemia caused by S. suis serotype 2. Capsular typing showed that 58% of the strains were nontypable. Of the 17 serotypes present, serotype 22 was the most prevalent. In the 7 farms without clinical signs on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, in less than 5% of the pigs by m-PCR or by bacteriologic culture. In the 8th farm, on which 2 pigs had clinical signs of septicemia on the day of sampling, we detected S. suis serotype 2 or 1/2, or both, by m-PCR in the tonsils of 40% of fattening pigs (21 wk old) that lacked symptoms. Molecular typing of the serotype 2 strains showed a common origin of contamination in these herds, given that 1 pattern (C1) was detected in the isolates from 6 of the 8 herds. However, up to 4 patterns were associated with septicemia and sudden death. Several patterns of S. suis serotype 2 can be responsible for disease in the same herd. These molecular tools may be useful for confident studies of the transmission of S. suis, thereby contributing to the control of S. suis infection.

Animals↗
PubMed2006

Phagocytosis and killing of Streptococcus suis by porcine neutrophils.

Streptococcus suis serotype 2 is an important swine pathogen responsible for diverse infections, mainly meningitis. Virulence factors and the pathogenesis of infection are not well understood. Neutrophils may play an important role in the pathogenesis of infection given that infiltration by neutrophils and mononuclear cells are frequently observed in lesions caused by S. suis. The objective of this work was to study the interactions between S. suis serotype 2 and porcine neutrophils. Results showed that suilysin is toxic to neutrophils and this could help S. suis evade innate immunity. Moreover, suilysin appears to affect complement-dependent killing by decreasing the opsonization of S. suis and the bactericidal capacity of neutrophils. Our results confirm that capsule polysaccharide protects S. suis against killing and phagocytosis by neutrophils. We also showed that the presence of specific IgG against S. suis serotype 2 promoted killing by neutrophils, indicating that the induction of a strong humoral response is beneficial for clearance of this pathogen.

Animals↗
PubMed2006

Haemophilus parasuis invades porcine brain microvascular endothelial cells.

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis.

Actins↗
PubMed2006

Proinflammatory cytokine and chemokine modulation by Streptococcus suis in a whole-blood culture system.

Streptococcus suis is an important swine and human pathogen. Inflammation, a hallmark of S. suis infection, is thought to be responsible for most clinical signs of meningitis, septicaemia and sudden death. In this work, using a porcine whole blood model, S. suis serotype 2 was shown to trigger the release of several pro-inflammatory cytokines as evaluated by reverse transcriptase-PCR and enzyme-linked immunosorbent assay. Although individual variations were observed among different S. suis strains, no correlations were observed between the strain origin/phenotype and cytokine levels. Live bacteria induced higher tumour necrosis factor alpha, interleukin-1 beta (IL-1beta) and IL-6 levels than did heat-killed bacteria. In contrast, heat-killed bacteria stimulated higher levels of IL-8 and monocyte chemotactic protein one (MCP-1). The bacterial cell wall was observed to be the major cytokine-inducting components, whereas capsule expression was important for MCP-1 activation. The presence of specific antibodies suppressed bacterial growth resulting in significantly reduced levels of cytokine production. Thus, antibody-mediated bacterial phagocytosis combined with suppressed inflammation may be beneficial for infection control strategies. We provide first evidence of S.suis-induction of pro-inflammatory swine cytokines and demonstrate the strength and relevance of the whole blood culture systems in the investigation of S. suis modulation of cytokine production.

Animals↗
PubMed2006

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay.

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.

Animals↗
PubMed2006

Identification of a surface protein of Streptococcus suis and evaluation of its immunogenic and protective capacity in pigs.

A Streptococcus suis surface protein reacting with convalescent-phase sera from pigs clinically infected by S. suis type 2 was identified. The apparent 110-kDa protein, designated Sao, exhibits typical features of membrane-anchored surface proteins of gram-positive bacteria, such as a signal sequence and an LPVTG membrane anchor motif. In spite of high identity with the partially sequenced genomes of S. suis Canadian strain 89/1591 and European strain P1/7, Sao does not share significant homology with other known sequences. However, a conserved avirulence domain that is often found in plant pathogens has been detected. Electron microscopy using an Sao-specific antiserum has confirmed the surface location of the Sao protein on S. suis. The Sao-specific antibody reacts with cell lysates of 28 of 33 S. suis serotypes and 25 of 26 serotype 2 isolates in immunoblots, suggesting its high conservation in S. suis species. The immunization of piglets with recombinant Sao elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are intratracheally challenged with a virulent strain in our conventional vaccination model.

Amino Acid Sequence↗
PubMed2006

Purified excreted-secreted antigens from Trypanosoma cruzi trypomastigotes as tools for diagnosis of Chagas' disease.

There is currently no "gold standard" test for the diagnosis of late-stage Chagas' disease. As a result, protection of the blood supply in areas where Chagas' disease is endemic remains problematic. A panel of 709 serum samples from subjects with confirmed Chagas' disease (n = 195), healthy controls (n = 400), and patients with other parasitic diseases (n = 114) was used to assess enzyme-linked immunosorbent assays (ELISAs) based on a concentrated extract of excretory-secretory antigens from either Brazil or Tulahuen strain Trypanosoma cruzi trypomastigotes (total trypomastigote excretory-secretory antigens [TESAs]). The total TESA-based assays had excellent overall sensitivity (100%) and specificity (>94%), except for cross-reactivity with Leishmania-infected sera. In an attempt to increase the specificity of the assay, immunoaffinity chromatography was used to purify the TESA proteins (TESA(IA) proteins). By Western blotting, a series of polypeptide bands with molecular masses ranging from 60 to 220 kDa were recognized by pooled sera positive for Chagas' disease. An ELISA based on TESA(IA) proteins had a slightly lower sensitivity (98.6%) but an improved specificity (100%) compared to the sensitivity and specificity of the total TESA protein-based ELISAs. A 60-kDa polypeptide was identified as a major contributor to the cross-reactivity with Leishmania. These data suggest the need for field validation studies of TESA- and TESA(IA)-based assays in regions where Chagas' disease is endemic.

Animals↗
PubMed2006

Streptococcus suis sequence type 7 outbreak, Sichuan, China.

An outbreak of Streptococcus suis serotype 2 emerged in the summer of 2005 in Sichuan Province, and sporadic infections occurred in 4 additional provinces of China. In total, 99 S. suis strains were isolated and analyzed in this study: 88 isolates from human patients and 11 from diseased pigs. We defined 98 of 99 isolates as pulse type I by using pulsed-field gel electrophoresis analysis of SmaI-digested chromosomal DNA. Furthermore, multilocus sequence typing classified 97 of 98 members of the pulse type I in the same sequence type (ST), ST-7. Isolates of ST-7 were more toxic to peripheral blood mononuclear cells than ST-1 strains. S. suis ST-7, the causative agent, was a single-locus variant of ST-1 with increased virulence. These findings strongly suggest that ST-7 is an emerging, highly virulent S. suis clone that caused the largest S. suis outbreak ever described.

Animals↗
PubMed2006

Molecular characterization of Streptococcus suis strains by 16S-23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis.

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.

Animals↗
PubMed2005

Upregulation of prostaglandin E2 and matrix metalloproteinase 9 production by human macrophage-like cells: synergistic effect of capsular material and cell wall from Streptococcus suis.

Streptococcus suis, an early colonizer of the upper respiratory tract, is a major swine pathogen worldwide that can cause meningitis, arthritis, pneumonia, and septicemia. While numerous studies on potential virulence factors of S. suis have been carried out over the past decade, the exact mechanisms by which this bacterium invades the host and migrates through the blood brain barrier (BBB) remain unclear. In the study presented here, we show that whole cells of S. suis were able to upregulate the production of prostaglandin E2 (PGE2) and matrix metalloproteinase 9 (MMP-9) by U937 human monoblastic cells differentiated into adherent macrophages in a dose- and time-dependent way. Capsular material and cell wall of S. suis were tested to determine which component was responsible for the induction of PGE2 and MMP-9 production by macrophages. The capsular material, even at low concentrations, strongly stimulated the production of PGE2 and MMP-9. While no stimulation was observed with the purified cell wall material, combining it with the capsular material resulted in a significant synergic effect on PGE2 and MMP-9 production. S. suis-mediated MMP-9 and PGE2 production by human macrophages may play a critical role in BBB disruption and tissue destruction.

Animals↗
PubMed2005

Truncation of the lipopolysaccharide outer core affects susceptibility to antimicrobial peptides and virulence of Actinobacillus pleuropneumoniae serotype 1.

We reported previously that the core oligosaccharide region of the lipopolysaccharide (LPS) is essential for optimal adhesion of Actinobacillus pleuropneumoniae, an important swine pathogen, to respiratory tract cells. Rough LPS and core LPS mutants of A. pleuropneumoniae serotype 1 were generated by using a mini-Tn10 transposon mutagenesis system. Here we performed a structural analysis of the oligosaccharide region of three core LPS mutants that still produce the same O-antigen by using methylation analyses and mass spectrometry. We also performed a kinetic study of proinflammatory cytokines production such as interleukin (IL)-6, tumor necrosis factor-alpha, IL1-beta, MCP-1, and IL8 by LPS-stimulated porcine alveolar macrophages, which showed that purified LPS of the parent strain, the rough LPS and core LPS mutants, had the same ability to stimulate the production of cytokines. Most interestingly, an in vitro susceptibility test of these LPS mutants to antimicrobial peptides showed that the three core LPS mutants were more susceptible to cationic peptides than both the rough LPS mutant and the wild type parent strain. Furthermore, experimental pig infections with these mutants revealed that the galactose (Gal I) and d,d-heptose (Hep IV) residues present in the outer core of A. pleuropneumoniae serotype 1 LPS are important for adhesion and overall virulence in the natural host, whereas deletion of the terminal GalNAc-Gal II disaccharide had no effect. Our data suggest that an intact core-lipid A region is required for optimal protection of A. pleuropneumoniae against cationic peptides and that deletion of specific residues in the outer LPS core results in the attenuation of the virulence of A. pleuropneumoniae serotype 1.

Actinobacillus pleuropneumoniae↗
PubMed2005

Acquisition of plasmin activity and induction of arachidonic acid release by Streptococcus suis in contact with human brain microvascular endothelial cells.

Infections caused by Streptococcus suis, a major swine pathogen, include meningitis, arthritis, pneumonia and septicaemia. In this study, we investigated interactions that may occur between human brain microvascular endothelial cells (HBMEC), the main constituent of the blood-brain barrier, and S. suis. We show that S. suis acquires plasmin activity in a time-dependent manner when in contact with cultured HBMEC. Cell-associated plasmin activity reached a plateau following a 48h co-incubation period. Zymography analysis revealed that HBMEC produce urokinase, which is probably involved in activation of plasminogen bound to S. suis. We also show that a S. suis culture supernatant which possesses both phospholipase C and haemolysin (suilysin) activities was able to induce the release of arachidonic acid from the membrane of HBMEC. Evidence suggests that the action of suilysin on HBMEC may be a prerequisite for the action of additional molecules such as phospholipase C. These new biological effects associated with S. suis may play an important role in the migration of S. suis through the blood-brain barrier and in the modulation of local inflammation.

Adult↗
PubMed2005

Biochemical analysis, cpn60 and 16S rDNA sequence data indicate that Streptococcus suis serotypes 32 and 34, isolated from pigs, are Streptococcus orisratti.

Streptococcus suis serotypes have traditionally been identified by morphology, biochemical profiling and serotyping. Analysis of the sequences of 16S rRNA and cpn60 genes of the 35 characterized serotypes of S. suis led to the observation that two serotypes 32 and 34, are significantly distinct from other S. suis serotypes and may represent a distinct species. Here we present DNA sequence data and biochemical profiles which indicate that S. suis serotypes 32 and 34, isolated from pigs, are clustered with Streptococcus orisratti, a Voges-Proskauer negative, alpha-haemolytic, aesculin-hydrolytic, Lancefield group A streptococcus isolated from the teeth of rats.

Animals↗
PubMed2005

Streptococcus suis serotype 2 binding to extracellular matrix proteins.

Streptococcus suis serotype 2 is a major swine and human pathogen that causes septicemia and meningitis. The ability of S. suis serotype 2 to bind to different extracellular matrix (ECM) proteins was evaluated by ELISA. All 23 strains tested bound to plasma and cellular fibronectin and collagen types I, III, and V, some to fibrin, vitronectin, and laminin, and none to the other ECM proteins tested. An unencapsulated isogenic mutant bound to ECM proteins better than its parental encapsulated strain, suggesting that the polysaccharide capsule interfered with binding. Cross-inhibition was observed between soluble plasma fibronectin and collagens in the ECM adherence assay, indicating that binding domains for both proteins exist on the same or nearby bacterial surface molecules. On the other hand, pre-incubation with plasma fibronectin increased binding to collagen IV, suggesting that S. suis might use fibronectin as a bridging molecule. The results of heat treatment and proteolytic digestion suggest that adhesins for these ECM proteins are proteinaceous in nature.

Animals↗
PubMed2005

Cloning, purification, and enzymatic properties of dipeptidyl peptidase IV from the swine pathogen Streptococcus suis.

In this study, the dipeptidyl peptidase IV (DPP IV) of the swine pathogen Streptococcus suis was cloned, overexpressed in Escherichia coli, and characterized. The coding region comprises 2,268 nucleotides containing an open reading frame that codes for a 755-amino-acid protein with a calculated molecular mass of 85 kDa. The amino acid sequence contained the sequence Gly-X-Ser-X-X-Gly, which is a consensus motif flanking the active-site serine shared by serine proteases. The recombinant DPP IV showed a high affinity for the synthetic peptide glycine-proline-p-nitroanilide and was strongly inhibited by Hg2+ and diprotin A.

Amino Acid Motifs↗
PubMed2005

Isolation of an atypical strain of Actinobacillus pleuropneumoniae serotype 1 with a truncated lipopolysaccharide outer core and no O-antigen.

A field isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine fibrinohemorrhagic necrotizing pleuropneumonia, was sent to the diagnostic laboratory for serotyping. The isolate presented a clear reaction, with both polyclonal antibodies against serotype 1 and monoclonal antibodies against the capsular polysaccharide of serotype 1. It also exhibited a PCR profile of Apx toxins expected for serotype 1. The isolate, however, failed to react with monoclonal antibodies against the O-antigen of serotype 1 lipopolysaccharide (LPS), suggesting a rough phenotype. The lipid A-core region of the isolate migrated faster than the corresponding region of the serotype 1 reference strain S4074 by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting the presence of a truncated core. Sugar analysis and mass spectrometry analysis of the O-deacylated LPS from the field isolate were consistent with the absence of O-antigen and truncation of the outer core compared to the wild-type reference strain. Experimental infection of pigs confirmed the virulence of the isolate. This is the first report of an isolate of A. pleuropneumoniae serotype 1 with a truncated outer core and a rough LPS phenotype. Veterinary diagnostic laboratories should be vigilant, since infections caused by such an isolate will not be detected by serological tests based on LPS O-antigen.

Actinobacillus Infections↗