Brachyspira in dogs: risk factors of shedding in central Germany and longitudinal study of an infected kennel.

BACKGROUND: Brachyspira (B.) pilosicoli is a zoonotic pathogen, able to infect different animal species such as pigs, poultry, and rodents, causing intestinal spirochetosis. An association of gastrointestinal clinical signs, such as diarrhea, with the isolation of B. pilosicoli from fecal samples or rectal swabs has not been proven in dogs. Other Brachyspira species commonly isolated from dogs, such as "B. canis" and "B. pulli", are considered commensals. This study investigated the occurrence of different Brachyspira species in rectal swabs and fecal samples in an independent canine cohort in central Germany. These included samples from shelter dogs, hunting dogs, and dogs presenting at regional small animal practices with various clinical signs. Data about the dogs, including potential risk factors for Brachyspira isolation, were obtained using a standardized questionnaire. The study also longitudinally investigated a colony of Beagle dogs for Brachyspira over 5 years. RESULTS: The rate of Brachyspira spp. isolation was 11% and included different Brachyspira species ("B. canis", "B. pulli", and B. pilosicoli). "B. canis" was detected in 18 dogs, whereas B. pilosicoli was only isolated from 1 dog in the independent cohort (not including the Beagle colony). Risk factors for shedding Brachyspira and "B. canis" were being less than 1 year of age and shelter origin. Gastrointestinal signs were not associated with the shedding of Brachyspira. B. pilosicoli and "B. canis" were isolated from several dogs of the same Beagle colony in 2017 and again in 2022, while Brachyspira was not isolated at multiple sampling time points in 2021. CONCLUSIONS: Shedding of B. pilosicoli in dogs appears to be uncommon in central Germany, suggesting a low risk of zoonotic transmission from dogs. Commensal status of "B. canis" and "B. pulli" is supported by the results of this study. Findings from the longitudinal investigation of the Beagle colony agree with an asymptomatic long-term colonization of dogs with "B. canis" and B. pilosicoli and suggest that introducing new animals in a pack can trigger an increased shedding of B. pilosicoli.

A critical review on experimental Streptococcus suis infection in pigs with a focus on clinical monitoring and refinement strategies.

Streptococcus suis (S. suis) is a major pig pathogen worldwide with zoonotic potential. Though different research groups have contributed to a better understanding of the pathogenesis of S. suis infections in recent years, there are still numerous neglected research topics requiring animal infection trials. Of note, animal experiments are crucial to develop a cross-protective vaccine which is highly needed in the field. Due to the severe clinical signs associated with S. suis pathologies such as meningitis and arthritis, implementation of refinement is very important to reduce pain and distress of experimentally infected pigs. This review highlights the great diversity of clinical signs and courses of disease after experimental S. suis pig infections. We review clinical read out parameters and refinement strategies in experimental S. suis pig infections published between 2000 and 2021. Currently, substantial differences exist in describing clinical monitoring and humane endpoints. Most of the reviewed studies set the body temperature threshold of fever as high as 40.5°C. Monitoring intervals vary mainly between daily, twice a day and three times a day. Only a few studies apply scoring systems. Published scoring systems are inconsistent in their inclusion of parameters such as body temperature, feeding behavior, and respiratory signs. Locomotion and central nervous system signs are more common clinical scoring parameters in different studies by various research groups. As the heterogenicity in clinical monitoring limits the comparability between studies we hope to initiate a discussion with this review leading to an agreement on clinical read out parameters and monitoring intervals among S. suis research groups.

A new S. suis serotype 3 infection model in pigs: lack of effect of buprenorphine treatment to reduce distress.

BACKGROUND: Streptoccocus suis (S. suis) is a major porcine pathogen causing meningitis, septicemia, arthritis and endocarditis. These diseases severely impair welfare of pigs. Experimental studies in pigs are important to better understand the pathogenesis and to identify protective antigens, as so far there is no vaccine available protecting against various serotypes (cps). Due to the severity of disease, application of appropriate refinement strategies in experimental S. suis infections is essential to reduce distress imposed on the piglets without jeopardizing the scientific output. The objectives of this study were to evaluate buprenorphine treatment as a refinement measure and serum cortisol levels as a distress read out parameter in a new S. suis cps3 infection model in pigs. RESULTS: Intravenous application of 2 × 108 CFU of S. suis cps3 (sly+, mrp+) to 6-week-old piglets led to severe morbidity in approximately 50% of the animals. Main pathological findings included suppurative meningoencephalitis and arthritis as well as fibrinosuppurative endocarditis. Buprenorphine treatment (0.05 mg/kg every 8 h) did not prevent signs of severe pain, high clinical scores, moderate to severe pathologies or high levels of serum cortisol in single severely affected piglets. Significant differences in the course of leukocytosis, induction of specific antibodies and bactericidal immunity were not recorded between groups with or w/o buprenorphine treatment. Of note, clinically unobtrusive piglets showed serum cortisol levels at 2 and 5 days post infectionem (dpi) comparable to the levels prior to infection with cps3. Cortisol levels in serum were significantly increased in piglets euthanized due to severe disease in comparison to clinically unobtrusive pigs. CONCLUSIONS: Different clinical courses and pathologies are induced after intravenous challenge of piglets with 2 × 108 CFU of this S. suis cps3 strain. The chosen protocol of buprenorphine application does not prevent severe distress in this infection model. Important parameters of the humoral immune response, such as the level of IgM binding to S. suis cps3, do not appear to be affected by buprenorphine treatment. Serum cortisol is a meaningful parameter to measure distress in piglets experimentally infected with S. suis and to evaluate refinement strategies. In this intravenous model, which includes close clinical monitoring and different humane endpoints, clinics and cortisol levels suggest convalescence in surviving piglets within 5 days following experimental infection.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

A critical review speculating on the protective efficacies of autogenous Streptococcus suis bacterins as used in Europe.

BACKGROUND: Streptococcus (S.) suis is a major porcine pathogen causing high morbidity worldwide. This includes well-managed herds with high hygiene standards. In Europe, no licensed vaccine is available. As practitioners are obliged to reduce the use of antibiotics, autogenous S. suis vaccines have become very popular in Europe. MAIN BODY: Autogenous vaccines (AV) are generally neither tested for safety, immunogenicity nor protective efficacy, which leads to substantial uncertainties regarding control of disease and return on investment. Here, S. suis publications are reviewed that include important data on epidemiology, pathologies and bacterin vaccination relevant for the use of AV in the field. Differences between herds such as the porcine reproductive and respiratory syndrome virus infection status and the impact of specific S. suis pathotypes are probably highly relevant for the outcome of immunoprophylaxis using autogenous S. suis bacterins. Thus, a profound diagnosis of the herd status is crucial for management of expectations and successful implementation of AV as a tool to control S. suis disease. Induction of opsonizing antibodies is an in vitro correlate of protective immunity elicited by S. suis bacterins. However, opsonophagocytosis assays are difficult to include in the portfolio of diagnostic services. CONCLUSION: Autogenous S. suis bacterins are associated with limitations and risks of failure, which can partly be managed through improvement of diagnostics.

Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro.

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG.

Vaccination with the immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis, leads to protection against a highly virulent serotype 9 strain.

Vaccination of weaning piglets with the recombinant IgM degrading enzyme of Streptococcus suis (S. suis), rIde Ssuis, elicits protection against disease caused by serotype (cps) 2 infection. In Europe, S. suis cps9 is at least as important as cps2 in causing severe herd problems associated with meningitis, septicemia and arthritis. The objective of this study was to determine humoral and cellular immunogenicities of rIde Ssuis suckling piglet vaccination and to investigate protection against a virulent cps9 strain. Vaccination in the 2nd and 4th week of life with rIde Ssuis and an oil-in-water adjuvant induced seroconversion against Ide Ssuis in 13 of 20 vaccinated piglets. In the 5th week, survival of the S. suis cps9 strain was significantly reduced in the blood of prime-booster vaccinated piglets. After a 2nd booster vaccination Ide Ssuis -reactive T helper (Th) cells partially producing TNF-α, IL-17A or IFN-É£ were detectable in rIde Ssuis -vaccinated but not in placebo-treated piglets and frequencies of Ide Ssuis -reactive Th cells correlated with α-Ide Ssuis-IgG levels. An intravenous challenge, conducted with a cps9 strain of sequence type (ST) 94, led to 89% mortality in placebo-treated piglets due to septicemia and meningitis. In contrast, all rIde Ssuis prime-booster-booster vaccinated littermates survived the challenge despite signs of disease such as fever and lameness. In conclusion, the described rIde Ssuis vaccination induces humoral and detectable Ide Ssuis -reactive Th cell responses and leads to protection against a highly virulent cps9 strain.

Comparing Cathelicidin Susceptibility of the Meningitis Pathogens Streptococcus suis and Escherichia coli in Culture Medium in Contrast to Porcine or Human Cerebrospinal Fluid.

Host defense peptides or antimicrobial peptides (AMPs), e.g., cathelicidins, have recently been discussed as a potential new treatment option against bacterial infections. To test the efficacy of AMPs, standardized methods that closely mimic the physiological conditions at the site of infection are still needed. The aim of our study was to test the meningitis-causing bacteria Streptococcus suis and Escherichia coli for their susceptibility to cathelicidins in culture medium versus cerebrospinal fluid (CSF). Susceptibility testing was performed in analogy to the broth microdilution method described by the Clinical and Laboratory Standard Institute (CLSI) to determine minimum inhibitory concentrations (MICs) of antimicrobial agents. MICs were determined using cation-adjusted Mueller-Hinton broth (CA-MHB), lysogeny broth (LB), Roswell Park Memorial Institute medium (RPMI) or Dulbecco's Modified Eagle's Medium (DMEM) (the latter two supplemented with 5% CA-MHB or blood) and compared with MICs obtained in porcine or human CSF. Our data showed that MICs obtained in CA-MHB as recommended by CLSI do not reflect the MICs obtained in the physiological body fluid CSF. However, the MICs of clinical isolates of S. suis tested in RPMI medium supplemented with CA-MHB, were similar to those of the same strains tested in CSF. In contrast, the MICs in the human CSF for the tested E. coli K1 strain were higher compared to the RPMI medium and showed even higher values than in CA-MHB. This highlights the need for susceptibility testing of AMPs in a medium that closely mimics the clinically relevant conditions.

The inflammatory response and neuronal injury in Streptococcus suis meningitis.

BACKGROUND: Many of the currently used models of bacterial meningitis have limitations due to direct inoculation of pathogens into the cerebrospinal fluid or brain and a relatively insensitive assessment of long-term sequelae. The present study evaluates the utility of a Streptococcus (S.) suis intranasal infection model for the investigation of experimental therapies in meningitis. METHODS: We examined the brains of 10 piglets with S. suis meningitis as well as 14 control piglets by histology, immunohistochemistry and in-situ tailing for morphological alterations in the hippocampal dentate gyrus and microglial activation in the neocortex. RESULTS: In piglets with meningitis, the density of apoptotic neurons was significantly higher than in control piglets. Moreover, scoring of microglial morphology revealed a significant activation of these cells during meningitis. The slight increase in the density of dividing cells, young neurons and microglia observed in piglets suffering from meningitis was not statistically significant, probably because of the short time frame between onset of clinical signs and organ sampling. CONCLUSIONS: The morphological changes found during S. suis meningitis are in accordance with abnormalities in other animal models and human autopsy cases. Therefore, the pig should be considered as a model for evaluating effects of experimental therapeutic approaches on neurological function in bacterial meningitis.

Differentiation of Brachyspira spp. isolated from laying hens using PCR-based methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Avian intestinal spirochetosis (AIS), an important but neglected disease in laying hens, is caused by Brachyspira pilosicoli, B. intermedia, and B. alvinipulli. Poultry are also frequently colonized by putatively nonpathogenic species such as B. murdochii and B. innocens. We evaluated the differentiation of Brachyspira species by 3 methods: sequencing of the reduced nicotinamide adenine dinucleotide (NADH) oxidase gene ( nox), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and a new multiplex (m)PCR targeting genes such as the tryptophanase A gene ( tnaA) and the p-aminobenzoyl-glutamate hydrolase subunit B gene ( abgB). Sequencing of 414 bp of the nox PCR amplification products generated from 41 pure cultures of avian Brachyspira isolates allowed presumptive species identification in 33 isolates with at least 99% identity in basic local alignment search tool analysis, including B. pilosicoli, B. intermedia, B. murdochii, B. innocens, and " B. pulli". MALDI-TOF MS analysis was found to be a reliable tool for differentiation after extension of the manufacturer's database. In the mPCR, all isolates identified as B. pilosicoli and B. intermedia were positive for abgB and tnaA, respectively. The mPCR might be very useful in detecting Brachyspira species in mixed cultures including not only nonpathogenic species, such as B. innocens, but also one of the AIS pathogens. We found that MALDI-TOF MS analysis combined with the mPCR targeting tnaA and abgB was suitable for the identification of avian isolates of B. pilosicoli and B. intermedia, 2 important agents of AIS.

Comparative analysis of clinics, pathologies and immune responses in BALB/c and C57BL/6 mice infected with Streptobacillus moniliformis.

Streptobacillus (S.) moniliformis is a rat-associated zoonotic pathogen that occasionally causes disease in other species. We investigated the working hypothesis that intranasal infection might lead to different immune responses in C57BL/6 and BALB/c mice associated with distinct pathologies. This study confirmed with 75% mortality the known high susceptibility of C57BL/6 mice to Streptobacillus moniliformis infection in comparison to BALB/c mice which did not develop signs of disease. Main pathologies in C57BL/6 mice were purulent to necrotizing lymphadenitis and pneumonia. Significant seroconversion was recorded in surviving mice of both strains. Differentiation of IgG-subclasses revealed mean ratios of IgG2b to IgG1 below 0.5 in sera of all mice prior to infection and of BALB/c mice post infection. In contrast, C57BL/6 mice had a mean IgG2b/IgG1 ratio of 2.5 post infection indicating a Th1 immune response in C57BL/6 versus a Th2 response in BALB/c mice. Evaluation of different sentinel systems revealed that cultural and serological investigations of these animals might not be sufficient to detect infection. In summary, an intranasal S. moniliformis infection model in C57BL/6 mice leading to purulent to necrotizing inflammations in the lung, the lymph nodes and other organs associated with a Th1 immune response is described.

Streptococcus suis serotype 9 endocarditis and subsequent severe meningitis in a growing pig despite specific bactericidal humoral immunity.

INTRODUCTION: . Meningitis and endocarditis are common pathologies of Streptococcussuis infections in pigs and humans. S. suis serotype 9 strains contribute substantially to health problems in European pig production, and immune prophylaxis against this serotype is very difficult. CASE PRESENTATION: . We report the clinical course and histopathological picture of a 10-week-old growing pig following experimental intravenous infection with S. suis serotype 9. The piglet showed rapid onset of severe clinical signs of meningitis 11 days post-intravenous challenge following prime-booster vaccination. Histopathological findings revealed a diffuse fibrinosuppurative leptomeningitis. Additionally, a polyphasic endocarditis valvularis thromboticans with numerous bacterial colonies was diagnosed. Bacteriological culture of the brain and the mitral valve confirmed association with the challenge strain. However, virulent serotype 2 and 9 strains were killed in the blood of this piglet ex vivo prior experimental infection. CONCLUSION: . Endocarditis induced by S. suis infection might develop and persist despite the presence of high specific bactericidal activity in the blood. Severe leptomeningitis is a putative sequela of such an endocarditis.

Pathogen-Reactive T Helper Cell Analysis in the Pig.

There is growing interest in studying host-pathogen interactions in human-relevant large animal models such as the pig. Despite the progress in developing immunological reagents for porcine T cell research, there is an urgent need to directly assess pathogen-specific T cells-an extremely rare population of cells, but of upmost importance in orchestrating the host immune response to a given pathogen. Here, we established that the activation marker CD154 (CD40L), known from human and mouse studies, identifies also porcine antigen-reactive CD4+ T lymphocytes. CD154 expression was upregulated early after antigen encounter and CD4+CD154+ antigen-reactive T cells coexpressed cytokines. Antigen-induced expansion and autologous restimulation enabled a time- and dose-resolved analysis of CD154 regulation and a significantly increased resolution in phenotypic profiling of antigen-responsive cells. CD154 expression identified T cells responding to staphylococcal Enterotoxin B superantigen stimulation as well as T cells responding to the fungus Candida albicans and T cells specific for a highly prevalent intestinal parasite, the nematode Ascaris suum during acute and trickle infection. Antigen-reactive T cells were further detected after immunization of pigs with a single recombinant bacterial antigen of Streptococcus suis only. Thus, our study offers new ways to study antigen-specific T lymphocytes in the pig and their contribution to host-pathogen interactions.

Identification of a novel host-specific IgG protease in Streptococcus phocae subsp. phocae.

Streptococcus (S.) phocae subsp. phocae causes bronchopneumonia and septicemia in a variety of marine mammals. Especially in harbor seals infected with phocine distemper virus it plays an important role as an opportunistic pathogen. This study was initiated by the detection of IgG cleavage products in Western blot analysis after incubation of bacterial supernatant with harbor seal serum. Hence, the objectives of this study were the identification and characterization of a secreted IgG cleaving protease in S. phocae subsp. phocae isolated from marine mammals. To further identify the responsible factor of IgG cleavage a protease inhibitor profile was generated. Inhibition of the IgG cleaving activity by iodoacetamide and Z-LVG-CHN2 indicated that a cysteine protease is involved. Moreover, an anti-IdeS antibody directed against the IgG endopeptidase IdeS of S. pyogenes showed cross reactivity with the putative IgG protease of S. phocae subsp. phocae. The IgG cleaving factor of S. phocae subsp. phocae was identified through an inverse PCR approach and designated IdeP (Immunoglobulin G degrading enzyme of S. phocae subsp. phocae) in analogy to the cysteine protease IdeS. Notably, recombinant (r) IdeP is a host and substrate specific protease as it cleaves IgG from grey and harbor seals but not IgG from harbor porpoises or non-marine mammals. The identification of IdeP represents the first description of a protein in S. phocae subsp. phocae involved in immune evasion. Furthermore, the fact that IdeP cleaves solely IgG of certain marine mammals reflects functional adaption of S. phocae subsp. phocae to grey and harbor seals as its main hosts.

Neutrophil extracellular trap formation in the Streptococcus suis-infected cerebrospinal fluid compartment.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

Identification and Characterization of IgdE, a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG.

Streptococcus suisis a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. ZoonoticS. suisinfections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease ofS. suisthat exclusively cleaves porcine IgM and represents the first virulence factor described, linkingS. suisto pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease ofS. suisthat exclusively targets porcine IgG. This enzyme, designated IgdE forimmunoglobulinG-degradingenzyme ofS. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that allS. suisstrains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressedin vivoduring infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.

The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is a highly protective antigen against serotype 2.

Streptococcus suis (S. suis) is a major porcine pathogen causing meningitis, arthritis and several other pathologies. Recently, we identified a highly specific immunoglobulin M degrading enzyme of S. suis, designated IdeSsuis, which is expressed by various serotypes. The objective of this work was to access the immunogenicity and protective efficacy of a recombinant vaccine including IdeSsuis. Vaccination with rIdeSsuis elicited antibodies efficiently neutralizing the IgM protease activity. Importantly, 18 piglets vaccinated with rIdeSsuis alone or in combination with bacterin priming were completely protected against mortality and severe morbidity after S. suis serotype 2 challenge. In contrast, 12 of the 17 piglets either treated with the placebo or primed with the bacterin only, succumbed to S. suis disease. Immunity against IdeSsuis was associated with increased killing of S. suis wt in porcine blood ex vivo leading to a tenfold difference in the bacterial survival factor in blood of placebo-treated and rIdeSsuis-vaccinated piglets. In conclusion, the results of this study indicate that rIdeSsuis is a highly protective antigen in pigs.

Identification of a novel DNase of Streptococcus suis (EndAsuis) important for neutrophil extracellular trap degradation during exponential growth.

The porcine and human pathogen Streptococcus suis induces and degrades neutrophil extracellular traps (NETs) in vitro. In this study, we investigated the working hypothesis that NET degradation is mediated not only by the known secreted S. suis nuclease A (SsnA) but also by a so-far undescribed putative endonuclease A of S. suis (designated EndAsuis) homologous to the pneumococcal endonuclease A (EndA). Comparative analysis was conducted to identify differences in localization, expression and function of EndAsuis and SsnA. In contrast to ssnA, endAsuis RNA expression was not substantially different during exponential and stationary growth. Modelling of the 3D structure confirmed a putative DRGH-motif-containing ßßα-metal finger catalytic core in EndAsuis. Accordingly, nuclease activity of recombinant EndAsuis with a point-mutated H165 was rescued through imidazol treatment. In accordance with a putative membrane anchor, nuclease activity caused by endAsuis was not detectable in the supernatant. Importantly, endAsuis determined nuclease activity of S. suis prominently during exponential growth. This activity depended on the presence of Mg(2+) but, in contrast to SsnA activity, not on Ca(2+). A pH of 5.4 did not inhibit endAsuis-encoded nuclease activity during exponential growth. NET degradation of S. suis harvested during exponential growth was significantly attenuated in the endAsuis mutant. In contrast to SsnA, mutagenesis of endAsuis did not result in a significantly higher susceptibility against the antimicrobial effect mediated by NETs. As degradation of bacterial DNA caused by S. suis depended on ssnA and endAsuis, further functions of both factors in the host-pathogen interaction might be envisioned.

Role of capsule and suilysin in mucosal infection of complement-deficient mice with Streptococcus suis.

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3(-/-) mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3(-/-) mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3(-/-) mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3(-/-) blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR(-/-) mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.

Streptococcus suis DNase SsnA contributes to degradation of neutrophil extracellular traps (NETs) and evasion of NET-mediated antimicrobial activity.

Streptococcus suis is an important cause of different pathologies in pigs and humans, most importantly fibrinosuppurative meningitis. Tissue infected with this pathogen is substantially infiltrated with neutrophils, but the function of neutrophil extracellular traps (NETs) - a more recently discovered antimicrobial strategy of neutrophils - in host defence against Strep. suis has not been investigated. The objective of this work was to investigate the interaction of Strep. suis with NETs in vitro. Strep. suis induced NET formation in porcine neutrophils and was entrapped but not killed by those NETs. As the amount of NETs decreased over time, we hypothesized that a known extracellular DNase of Strep. suis degrades NETs. Though this nuclease was originally designated Strep. suis-secreted nuclease A (SsnA), this work demonstrated surface association in accordance with an LPXTG cell wall anchor motif and partial release into the supernatant. Confirming our hypothesis, an isogenic ssnA mutant was significantly attenuated in NET degradation and in protection against the antimicrobial activity of NETs as determined in assays with phorbol myristate acetate (PMA)-stimulated human neutrophils. Though assays with PMA-stimulated porcine neutrophils suggested that SsnA also degrades porcine NETs, phenotypic differences between wt and the isogenic ssnA mutant were less distinct. As SsnA expression was crucial for neither growth in vitro nor for survival in porcine or human blood, the results indicated that SsnA is the first specific NET evasion factor to be identified in Strep. suis.