Immune response induced by a Streptococcus suis multi-serotype autogenous vaccine used in sows to protect post-weaned piglets.

Streptococcus suis is a bacterial pathogen that causes important economic losses to the swine industry worldwide. Since there are no current commercial vaccines, the use of autogenous vaccines applied to gilts/sows to enhance transfer of passive immunity is an attractive alternative to protect weaned piglets. However, there is no universal standardization in the production of autogenous vaccines and the vaccine formulation may be highly different among licenced manufacturing laboratories. In the present study, an autogenous vaccine that included S. suis serotypes 2, 1/2, 5, 7 and 14 was prepared by a licensed laboratory and administrated to gilts using a three-dose program prior to farrowing. The antibody response in gilts as well as the passive transfer of antibodies to piglets was then evaluated. In divergence with previously published data with an autogenous vaccine produced by a different company, the increased response seen in gilts was sufficient to improve maternal antibody transfer to piglets up to 5 weeks of age. However, piglets would still remain susceptible to S. suis disease which often appears during the second part of the nursery period. Vaccination did not affect the shedding of S. suis (as well as that of the specific S. suis serotypes included in the vaccine) by either gilts or piglets. Although all antibiotic treatments were absent during the trial, the clinical protective effect of the vaccination program with the autogenous vaccine could not be evaluated, since limited S. suis cases were present during the trial, confirming the need for a complete evaluation of the clinical protection that must include laboratory confirmation of the aetiological agent involved in the presence of S. suis-associated clinical signs. Further studies to evaluate the usefulness of gilt/sow vaccination with autogenous vaccines to protect nursery piglets should be done.

Long-chain LPS-based enzyme-linked immunosorbent assay to detect swine herds infected by Actinobacillus pleuropneumoniae serotype 17.

Actinobacillus pleuropneumoniae serotype 17, one of the two most recent serotypes described, has been isolated from diseased pigs in North America. Yet, no serological test for surveillance has been developed so far. An enzyme-linked immunosorbent assay (ELISA) using the long-chain lipopolysaccharide antigen (LC-LPS) of this serotype is described. As predicted by previous genetic data on the O-antigen locus, cross reactions were observed between this serotype and serotypes 3, 6, 8, and 15. Although animals infected by serotype 17 would be detected using the current serotype 3 LC-LPS ELISA, better results may be obtained when plates are coated with the antigen purified from the homologous serotype.

Développement d'un test sérologique ELISA en utilisant l'antigène de la chaine O du LPS afin de détecter les troupeaux infectés par Actinobacillus pleuropneumoniae sérotype 17. Le récemment décrit Actinobacillus pleuropneumoniae sérotype 17 a été isolé chez des porcs malades en Amérique du Nord. Pourtant, aucun test sérologique n'a été mis au point à ce jour. Nous avons développé un test ELISA utilisant l'antigène lipopolysaccharidique à longue chaîne (LC-LPS) de ce sérotype. Tel que prévu par l'analyse du locus de l'antigène O du LPS, des réactions croisées ont été observées entre ce sérotype et les sérotypes 3, 6, 8 et 15. Bien que les animaux infectés par le sérotype 17 pourraient être détectés en utilisant le sérotype 3 LC-LPS ELISA, plus de sensibilité pourrait être obtenue en utilisant l'antigène purifié à partir du sérotype homologue.(Traduit par les auteurs).

Serotype-specific role of antigen I/II in the initial steps of the pathogenesis of the infection caused by Streptococcus suis.

Streptococcus suis is one of the most important post-weaning porcine bacterial pathogens worldwide. The serotypes 2 and 9 are often considered the most virulent and prevalent serotypes involved in swine infections, especially in Europe. However, knowledge of the bacterial factors involved in the first steps of the pathogenesis of the infection remains scarce. In several pathogenic streptococci, expression of multimodal adhesion proteins known as antigen I/II (AgI/II) have been linked with persistence in the upper respiratory tract and the oral cavity, as well as with bacterial dissemination. Herein, we report expression of these immunostimulatory factors by S. suis serotype 2 and 9 strains and that AgI/II-encoding genes are carried by integrative and conjugative elements. Using mutagenesis and different in vitro assays, we demonstrate that the contribution of AgI/II to the virulence of the serotype 2 strain used herein appears to be modest. In contrast, data demonstrate that the serotype 9 AgI/II participates in self-aggregation, induces salivary glycoprotein 340-related aggregation, contributes to biofilm formation and increased strain resistance to low pH, as well as in bacterial adhesion to extracellular matrix proteins and epithelial cells. Moreover, the use of a porcine infection model revealed that AgI/II contributes to colonization of the upper respiratory tract of pigs. Taken together, these findings suggest that surface exposed AgI/II likely play a key role in the first steps of the pathogenesis of the S. suis serotype 9 infection.

Determining Streptococcus suis serotype from short-read whole-genome sequencing data.

BACKGROUND: Streptococcus suis is divided into 29 serotypes based on a serological reaction against the capsular polysaccharide (CPS). Multiplex PCR tests targeting the cps locus are also used to determine S. suis serotypes, but they cannot differentiate between serotypes 1 and 14, and between serotypes 2 and 1/2. Here, we developed a pipeline permitting in silico serotype determination from whole-genome sequencing (WGS) short-read data that can readily identify all 29 S. suis serotypes. RESULTS: We sequenced the genomes of 121 strains representing all 29 known S. suis serotypes. We next combined available software into an automated pipeline permitting in silico serotyping of strains by differential alignment of short-read sequencing data to a custom S. suis cps loci database. Strains of serotype pairs 1 and 14, and 2 and 1/2 could be differentiated by a missense mutation in the cpsK gene. We report a 99 % match between coagglutination- and pipeline-determined serotypes for strains in our collection. We used 375 additional S. suis genomes downloaded from the NCBI's Sequence Read Archive (SRA) to validate the pipeline. Validation with SRA WGS data resulted in a 92 % match. Included pipeline subroutines permitted us to assess strain virulence marker content and obtain multilocus sequence typing directly from WGS data. CONCLUSIONS: Our pipeline permits rapid and accurate determination of S. suis serotype, and other lineage information, directly from WGS data. By discriminating between serotypes 1 and 14, and between serotypes 2 and 1/2, our approach solves a three-decade longstanding S. suis typing issue.

The protective protein Sao (surface antigen one) is not a critical virulence factor for Streptococcus suis serotype 2.

Surface antigen one (Sao) is protein that was shown to protect pigs in vaccination trials against disease caused by Streptococcus suis. Here, we used an isogenic Δsao mutant to investigate the insofar unknown role of Sao in S. suis virulence. The Δsao mutant and its wild-type parent strain (sequence type 1, European strain) did not present significant differences on levels of adhesion/invasion of swine respiratory epithelial cells. Moreover, the Δsao mutant resisted phagocytosis by macrophages similarly to the parent strain and it was still virulent in a mouse model of infection. Our findings suggest a minor role of Sao in S. suis serotype 2 virulence.

Invasive Bacterial Infections of the Musculoskeletal and Central Nervous System during Pig Rearing: Detection Frequencies of Different Pathogens and Specific Streptococcus suis Genotypes.

Locomotor and central nervous system disorders occur during pig rearing, but there is no systematic recording of the different causative agents in Germany. Joint and meningeal swabs, kidneys, lungs, and eight different lymph nodes per pig were cultured, and isolated pathogens were identified using polymerase chain reactions (PCRs). The cps and pathotype of Streptococcus suis (S. suis) isolates were determined using multiplex-PCR. S. suis was the most important pathogen in the infected joints (70.8%) and meningeal swabs (85.4%) and was most frequently detected in both sites in suckling and weaning piglets. To elucidate the possible portal of entry of S. suis, eight different lymph nodes from 201 pigs were examined in a prospective study. S. suis was detected in all examined lymph nodes (n = 1569), including the mesenteric lymph nodes (15.8%; n = 121/765), with cps 9 (37.2%; n = 147) and cps 2 (24.3%; n = 96) being the most dominating cps types. In piglets with a systemic S. suis infection, different lymph nodes are frequently infected with the invasive S. suis strain, which does not help clarify the portal of entry for S. suis.

Interactions of Mycoplasma hyopneumoniae and/or Mycoplasma hyorhinis with Streptococcus suis Serotype 2 Using In Vitro Co-Infection Models with Swine Cells.

Bacterial and/or viral co-infections are very common in swine production and cause severe economic losses. Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and Streptococcus suis are pathogenic bacteria that may be found simultaneously in the respiratory tracts of pigs. In the present study, the interactions of S. suis with epithelial and phagocytic cells in the presence or absence of a pre-infection with M. hyopneumoniae and/or M. hyorhinis were studied. Results showed relatively limited interactions between these pathogens. A previous infection with one or both mycoplasmas did not influence the adhesion or invasion properties of S. suis in epithelial cells or its resistance to phagocytosis (including intracellular survival) by macrophages and dendritic cells. The most important effect observed during the co-infection was a clear increment in toxicity for the cells. An increase in the relative expression of the pro-inflammatory cytokines IL-6 and CXCL8 was also observed; however, this was the consequence of an additive effect due to the presence of different pathogens rather than a synergic effect. It may be hypothesized that if one or both mycoplasmas are present along with S. suis in the lower respiratory tract at the same time, then increased damage to epithelial cells and phagocytes, as well as an increased release of pro-inflammatory cytokines, may eventually enhance the invasive properties of S. suis. However, more studies should be carried out to confirm this hypothesis.

Distribution and characterization of Streptococcus suis serotypes isolated from January 2015 to June 2020 from diseased pigs in Québec, Canada.

Streptococcus suis is one of the most important swine bacterial pathogens causing economic losses. This report presents the serotype distribution of S. suis recovered from diseased pigs in Québec from January 2015 to June 2020. Serotypes 1/2 and 2 predominated, followed by serotypes 7, 3, 5, 4, 9, 1, and 14. Compared to previously reported data, very few changes could be observed concerning the serotype distribution, indicating a relative stability. Half of the untypable isolates did not belong to the species S. suis sensu stricto, as determined by recN polymerase chain reaction. Less than 10% of "real S. suis" isolates were untypable. The genetic diversity of S. suis serotypes 1, 2, and 14, as analyzed by multilocus sequence typing, was mainly represented by sequence type (ST)1, ST28, ST25, and ST94. All ST1 isolates (considered highly virulent) belonged to either serotype 1 or 14.

Streptococcus suis est l'un des agents pathogènes bactériens porcins les plus importants et qui cause d'importantes pertes économiques. Ce rapport présente la distribution des sérotypes isolés à partir de porcs malades au Québec de janvier 2015 à juin 2020. Les sérotypes 1/2 et 2 prédominent, suivis des sérotypes 7, 3, 5, 4, 9, 1, et 14. Peu de changements par rapport aux années précédentes ont pu être observés en ce qui concerne la distribution des sérotypes. Plus de la moitié des isolats n'appartenaient pas à l'espèce S. suis sensu stricto, selon la réaction en chaîne par polymérase recN. Moins de 10 % des isolats des « vrais ¼ S. suis étaient non-typables. La diversité génétique des sérotypes 1, 2, et 14 de S. suis, telle qu'analysée par typage de séquences multilocus, était principalement représentée par les types de séquence (ST)1, ST28, ST25 et ST94. Tous les isolats ST1 (considérés comme hautement virulents) appartenaient aux sérotypes 1 ou 14.(Traduit par les auteurs).

Production and characterization of a murine anti-dal monoclonal antibody for blood typing in dogs.

Considering the strong immunogenicity of the Dal antigen, and that > 98% of dogs, including blood donors, are Dal-positive, finding compatible blood for a previously transfused Dal-negative patient may be challenging. This is exacerbated by limited access to typing reagents, which currently rely on polyclonal antibodies (PAb) produced following sensitization of dogs. Therefore, the objective of this study was to produce and characterize an anti-Dal murine monoclonal antibody (MAb). Conventional hybridoma technology was used to produce MAb directed against canine red blood cells (cRBC). Briefly, female BALB/c mice were immunized via repeated intraperitoneal injections of washed Dal-positive cRBC (DEA 1,3,7 negative; DEA 4,5 positive) until serologic titers were sufficient (>1:1000). Following fusion with myeloma cells, 573 hybridoma cell culture supernatants were obtained and screened for MAb of interest using a gel column agglutination technique and known Dal-negative and Dal-positive cRBC. Fifteen supernatants led to cRBC agglutination, but only one had the desired pattern (i.e. anti-Dal). To assess its specificity and sensitivity, Dal blood typing of 62 canine EDTA-blood samples was performed using the anti-Dal MAb and two canine PAb: 45 Dal-positive and 17 Dal-negative were identified with 100% agreement between reagents (kappa =1). The anti-Dal MAb produced was further determined to be an IgG1. Conventional hybridoma technology, aided by a gel column technique, has enabled the production of a murine MAb specific against the canine Dal antigen. This will ensure long-term perennity of Dal blood typing, facilitate clinical management and research, as well as avoid resorting to repeat dog sensitization.

Lineage and virulence of Streptococcus suis serotype 2 isolates from North America.

We performed multilocus sequence typing of 64 North American Streptococcus suis serotype 2 porcine isolates. Strains were sequence type (ST) 28 (51%), ST25 (44%), and ST1 (5%). We identified nonrandom associations between STs and expression of the virulence markers suilysin (SLY), muramidase-relased protein (MRP), and extracellular factor (EF). Expression of pili encoded by the srtF and srtG pilus clusters was also nonrandomly associated with STs. ST1 strains were SLY+ EF+ MRP+ srtF pilus+ srtG pilus-. ST25 strains were SLY- EF- MRP- srtF pilus- srtG pilus+, and most ST28 strains were SLY- MRP+ EF- srtF pilus+ srtG pilus+. ST28 isolates proved essentially nonvirulent in a mouse infection model; ST25 strains showed moderate virulence and ST1 isolates were highly virulent. ST1 is responsible for a high proportion of S. suis disease in humans worldwide. Its presence in North America indicates that potential zoonotic S. suis outbreaks in this continent cannot be disregarded.

Rapid Detection and Typing of Actinobacillus pleuropneumoniae Serovars Directly From Clinical Samples: Combining FTA® Card Technology With Multiplex PCR.

Actinobacillus pleuropneumoniae (APP), the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality, and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs. We have optimized the processing of 3-mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-multiplex PCR (mPCR) assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.

Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars.

Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-013 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13-14 and 16-19.

Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation.

We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.

Structure determination of Streptococcus suis serotype 2 capsular polysaccharide.

The capsular polysaccharide (CPS) of Streptococcus suis serotype 2 was isolated, purified, chemically modified, and characterized. Sugar and absolute configuration analyses of the CPS gave the following composition: D-Gal, 3; D-Glc, 1; D-GlcNAc, 1; D-Neu5Ac, 1; L-Rha, 1. Sialic acid was found to be terminal, and the CPS was quantitatively desialylated by mild acid hydrolysis. The CPS was also submitted to periodate oxidation followed by borohydride reduction and Smith degradation. Sugar and methylation analysis, 1H and 13C nuclear magnetic resonance, and mass spectrometry of the native CPS or of its specifically modified products allowed to determine the repeating unit sequence: [4)[Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)]Gal(beta1-4)[Gal(alpha1-3)]Rha(beta1-4)Glc(beta1-]n. The backbone sequence was found to be identical to that of Streptococcus agalactiae or group B Streptococcus (GBS) type VIII and Streptococcus pneumoniae type 23F. The S. suis CPS shares the sequence Neu5Ac-Gal-GlcNAc-Gal in common with GBS types Ia, Ib, II, III, and IV CPSs but differs from them by the presence of rhamnose and the fact that sialic acid is 2,6- rather than 2,3-linked to the following Gal. A correlation between the S. suis CPS sequence and genes of the serotype 2 cps locus encoding putative enzymes responsible for the biosynthesis of the repeating unit was tentatively established.

Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.