Formate-tetrahydrofolate ligase is involved in the virulence of Streptococcus suis serotype 2
2016, Microbial Pathogenesis
Citation Excerpt :
The murine infection model has been widely used for the assessment of S. suis virulence [29–32]. However, it was reported that murine models are unsuitable for extrapolation to S. suis infection in pigs, as the virulence of S. suis 2 in mice and pigs is host-specific . To further confirm the involvement of formate-tetrahydrofolate ligase in S. suis 2 virulence, we conducted a competitive-infection assay in piglets, which are the natural hosts of S. suis.
Streptococcus suis is an emerging zoonotic pathogen that causes severe infections in pigs and humans. However, the pathogenesis of S.suis remains unclear. The present study targeted a putative virulence-associated factor (fhs, encoding the formate-tetrahydrofolate ligase) of S.suis. To investigate the role of fhs in the virulence potential of S.suis serotype 2, an fhs deletion mutant (Δfhs) and the corresponding complementation strain (CΔfhs) were generated. The Δfhs mutant displayed similar growth compared to that of the wild-type and complementation strains. Using murine and pig infection models, we demonstrated for the first time that the formate-tetrahydrofolate ligase is required for the full virulence of S.suis 2. Our findings provide a new insight into the pathogenesis of S.suis 2.
Characterization of the immune response and evaluation of the protective capacity of rSsnA against Streptococcus suis infection in pigs
2016, Comparative Immunology, Microbiology and Infectious Diseases
Citation Excerpt :
This protein combined with aluminium hydroxide (ALOH) presented a good protective capacity against a challenge with the homologous strain of S. suis SS2 in mouse model . However, different authors have questioned whether the results obtained in murine models can be applied to pigs [25,26]. Therefore, the aim of this study was to determine the efficacy of the recombinant SsnA protein (rSsnA) as vaccine candidate combined with ALOH plus Quil A as adjuvants against a challenge with the homologous strain of S. suis SS2 in a pig model of infection.
The efforts made to develop vaccines against Streptococcus suis have failed because of lack of common antigens cross-reactive against different serotypes of this species. The cell wall-anchored proteins can be good vaccine candidates due to their high expression and accessibility to antibodies, among these, a cell-wall protein, DNA-nuclease (SsnA), present in most of the S. suis serotypes and clinical isolates collected from infected pigs, was selected. An experimental challenge against S. suis serotype 2 in a pig model was used to validate the efficacy of recombinant SsnA combined with aluminium hydroxide plus Quil A as adjuvants, previously tested in mice by our research group with good results. In our study, clinical characteristics, bacterial load and spread, haematological and immunological parameters and the antibody response, including the opsonophagocytosis analysis of the sera were evaluated. Moreover the composition of peripheral blood leukocyte populations was studied in infected animals. The results show that the immunization of piglets with rSsnA elicits a significant humoral antibody response. However, the antibody response is not reflected in protection of pigs that are challenged with a virulent strain in our conventional vaccination model. Further studies are necessary to evaluate the use of rSsnA as a vaccine candidate for swine.
Assessment of the pathogenesis of Streptococcus suis type 2 infection in piglets for understanding streptococcal toxic shock-like syndrome, meningitis, and sequelae
2014, Veterinary Microbiology
Citation Excerpt :
In addition, likely due to the lack of a suitable animal model, the pathogenesis of the disease progressing into STSLS or meningitis, as well as the sequelae, caused by the Chinese virulent ST7 strain has not been systematically elucidated. As the natural host, pigs are the best model to study S. suis infection and to reflect the symptoms of patients more exactly than other animal models (e.g., mouse) (Vecht et al., 1997). Thus, to gain insight into the pathogenesis of the entire course of the disease, piglets were used as disease model for ST7 infection.
Streptococcus suis type 2 (SS2) is an zoonotic pathogen that had caused outbreaks in 1998 and 2005 in China. It is still not very clear how the disease progresses into the streptococcal toxic shock-like syndrome (STSLS) or meningitis, as well as the sequelae from the survivals. The present study used piglets as infection model to systematically investigate the pathogenesis of the infection caused by the SS2 strain 05ZYH33. The infected piglets showed joint swelling, lameness, and crouch at beginning, then developed into septic-like shock syndrome (SLSS) or prostration syndrome, at last the survivals showed physical activity impairment. The morbidity and mortality were 100% (71% for SLSS, 29% for prostration syndrome) and 29%, respectively. The pigs exhibiting SLSS had deep invasive infections in tissues and organs, and displayed more severe bacteremia and cytokine secretion in the bloodstream and organs than pigs with prostration syndrome. Moreover, the polymorphisms in the toll-like receptor 1 (TLR1) and TLR2 genes varied between the pigs affected with SLSS and prostration syndrome. Several lines of evidence indicated that SS2 infection progression into SLSS or relatively lighter prostration syndrome in pigs is closely related to the degrees of bacteremia and cytokine storm, which may be inherently determined by the diversity of innate immunity-associated genes. Furthermore, brain lesions, such as venous thrombosis, may directly contribute to the sequelae in human cases, were identified in the pigs. These results might help us to further understand the pathogenesis of SS2 in humans.
Polysaccharide capsule and suilysin contribute to extracellular survival of Streptococcus suis co-cultivated with primary porcine phagocytes
2008, Veterinary Microbiology
Streptococcus suis is a major cause of meningitis, sepsis and arthritis in piglets and a zoonotic agent. Survival in the blood circulation system represents a major step in pathogenesis of S. suis infections. To get further insights into the mechanisms of S. suis survival in the host, we compared a highly virulent S. suis serotype 2 strain with its non-encapsulated and suilysin-deficient mutants in their abilities to resist phagocytosis and killing by polymorphonuclear neutrophils (PMNs) and mononuclear cells. PMNs displayed a higher capacity to take up encapsulated bacteria than mononuclear cells, whereas both cell types internalized efficiently non-encapsulated S. suis. Differentiation of extracellular and intracellular survival of the WT strain revealed that in PMNs the majority of the cell-associated streptococci were intracellular, whereas in mononuclear cells the majority remained attached to the cell surface. S. suis survived mainly extracellularly, since both cells killed intracellular bacteria to a similar extent. As a consequence of different resistance to phagocytosis, only the encapsulated S. suis strains survived co-cultivation with PMNs. Comparison of the WT strain with its encapsulated suilysin-deficient mutant revealed reduced survival of the mutant after co-cultivation with PMNs. Involvement of suilysin in inhibition of phagocytosis was further confirmed by the use of anti-suilysin antibodies and recombinant suilysin. Kinetic experiments with PMNs suggested that reduced survival of the mutant strain was mainly associated with an increased uptake, whilst both strains adhered similarly. Concluding, our results indicate that the capsule and the suilysin play important roles in S. suis survival in the host by interfering with phagocytic uptake.
Role of suilysin in pathogenesis of Streptococcus suis capsular serotype 2
2003, Microbial Pathogenesis
Three suilysin (SLY) knockout mutant strains of Streptococcus suis serotype 2 were generated by allelic replacement from one North American and two European wild type strains. The mutants were characterized by Southern blot, Western blot and phenotyping. In vitro bactericidal testing showed that both wild type and SLY mutants were resistant to bactericidal factors in whole pig blood. To demonstrate the role of SLY during S. suis infection, four animal trials were carried out using young pigs. Either high dose (4×106CFU/ml/pig) or low dose (0.5×106CFU/ml/pig) live cell aerosol was applied to the pharynx. In one trial, a low challenge dose of North American strain SX332 and its isogenic sly− mutant strain (SX932) resulted in acute disease in 3/5 of pigs exposed to the wild type strain, while 5/5 of pigs exposed to the mutant strain survived the trial. In the repeat trial, 1/8 of pigs in wild type group and 6/8 of pigs in mutant group developed disease. The high dose trial with 332/932 pair showed that 4/8 pigs challenged with wild type and 5/8 of pigs challenged with mutant strain developed disease respectively. The third low dose trial, using European strain 31533 and its isogenic sly− mutant strain SX911, showed that 1/8 of pigs challenged with the wild type strain and 4/8 of pigs challenged with the corresponding mutant strain developed disease. All the diseased pigs showed fever, clinical signs and developed septicemia. S. suis was isolated from tissue samples such as brain, submandibular lymph node, lung, spleen, liver, heart or joint. Serum antibody titer against cell surface proteins changed little while the antibody titer against SLY increased only in the wild type group after challenge. sly gene was cloned and expressed in E. coli. The recombinant SLY (rSLY) protein showed 800 hemolysin units per μg protein. In vitro study showed that rSLY triggered TNFα production by human monocytes and IL-6 production by pig pulmonary alveolar macrophages and monocytes. Thus, the results of this study suggest that SLY does not seem to be a critical virulence factor for S. suis serotype 2 respiratory infection, but by stimulating cytokine release it may play a role in innate immunity.
Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2
2001, Veterinary Microbiology
The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6ml of antiserum the day before challenge and four pigs used as challenge controls. Pigs were infected by subcutaneous injection with approximately 1011 colony forming units of S. suis serotype 2. Clinical disease in the pigs that could be attributed to infection with S. suis was reduced from 88 to 35% (P=0.015). The percentage of pigs with lesions that could be associated with S. suis was reduced from 88 to 22% (P=0.002) and isolation of S. suis serotype 2 was reduced from five (63%) out of eight pigs in the combined challenge control groups to 3 (13%) out of 23 pigs in the combined treatment groups. These results indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs.
Characterisation of Streptococcus suis isolates from wild boars (Sus scrofa)
The Veterinary Journal, Volume 200, Issue 3, 2014, pp. 464-467
Wild boar are widely distributed throughout the Iberian Peninsula and can carry potentially virulent strains of Streptococcus suis. The objective of this study was to determine the prevalence of S.suis in wild boars from two large geographical regions of Spain. Serotypes 1, 2, 7 and 9 identified were further genetically characterised by virulence-associated genotyping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to determine the population structure of S.suis carried by these animals. Streptococcus suis was isolated from 39.1% of the wild boars examined: serotype 9 was the most frequently isolated (12.5%), followed by serotype 1 (2.5%). Serotype 2 was rarely isolated (0.3%). Eighteen additional serotypes were identified indicating wide diversity of this pathogen within the wild boar population. This heterogeneity was confirmed by PFGE and MLST analyses and the majority of isolates exhibited the virulence-associated genotype mrp-/epf-/sly-. The results of this study highlight that the carriage of S.suis by wild boars is commonplace. However, MLST data indicate that these isolates are not related to prevalent clonal complexes ST1, ST16, ST61 and ST87 typically associated with infection of pigs or humans in Europe.
Characterization of IgA1 protease as a surface protective antigen of Streptococcus suis serotype 2
Microbes and Infection, Volume 18, Issue 4, 2016, pp. 285-289
IgA1 protease of Streptococcus suis serotype 2 (SS2) has been proven to be relative with virulence and immunogenicity, however, its protective efficacy remained to be evaluated. The present study found evidence that immunization with purified recombinant IgA1 protease (600-1926aa) could induce high IgG antibody titers and could confer complete protection against a challenge with a lethal dose of SS2 in a mouse model. In addition, our findings confirmed that the IgA1 protease distributes on the surface of SS2. Therefore, the present study identified the virulence-associated protein, IgA1 protease, as a novel surface protective antigen of SS2.
Monitoring of antimicrobial susceptibility of Streptococcus suis in the Netherlands, 2013–2015
Veterinary Microbiology, Volume 194, 2016, pp. 5-10
The objective of the present study was to analyse the in vitro antimicrobial susceptibility of Streptococcus suis isolates from post-mortem samples from pigs in the Netherlands. S. suis isolates originated from diagnostic submissions of pigs sent to the Pathology Department of GD Animal Health, from April 2013 till June 2015. Minimal inhibitory concentrations (MICs) of in total 15 antimicrobials were assessed by broth microdilution following CLSI recommendations. MIC50 and MIC90 values were determined and MICs were interpreted as susceptible, intermediate and resistant using CLSI veterinary breakpoints (when available). Emergence of resistance among S. suis (n=1163) derived from clinical submissions of pigs appeared to be limited. Resistance to ampicillin, ceftiofur, clindamycin, enrofloxacin, florfenicol, penicillin, trimethoprim/sulfamethoxazole and tetracycline was 0.3%, 0.5%, 48.1%, 0.6%, 0.1%, 0.5%, 3.0%, and 78.4%, respectively. Cross-resistance between penicillin and ampicillin appeared to be incomplete. MIC values of erythromycin, clindamycin, neomycin, penicillin and tilmicosin for isolates originating from grower/finisher pigs were significantly more often lower than the MIC values of isolates from suckling/weaned piglets. It has to be kept in mind that these results represent only part of the Dutch pig population and it can be discussed whether this is a representative sample. Interpretation of the MIC results of (clinically relevant) antimicrobials tested for treatment of S. suis infection is strongly hampered by the lack of CLSI-defined veterinary clinical breakpoints that are animal species- and body site-specific. Therefore, and to conduct a clinically reliable monitoring of antimicrobial susceptibility of veterinary pathogens, more species- and organ-specific veterinary breakpoints are urgently needed.
Screening of virulence-associated genes as a molecular typing method for characterization of Streptococcus suis isolates recovered from wild boars and pigs
The Veterinary Journal, Volume 209, 2016, pp. 108-112
Streptococcus suis is an important zoonotic pathogen associated with a wide range of diseases in pigs, but has also been isolated from wild animals such as rabbits and wild boars. In the current study, 126 S.suis isolates recovered from pigs (n = 85) and wild boars (n = 41) were tested by polymerase chain reaction (PCR) for the presence of nine virulence-associated genes. S.suis isolates from wild boars were differentiated by the lower detection rates of the epf, sly, mrp, sao and dltA genes (0%, 2.4%, 2.4%, 4.8% and 21.9%, respectively) compared with the isolates from pigs (56.5%, 75.3%, 56.5%, 88.2.0% and 88.2%, respectively). The differences in the content of these virulence-associated genes were statistically significant (P < 0.05). There was a correlation between the variants saoM and saoL and serotypes 2 and 9, respectively (P < 0.05). Isolates were classified into 31 virulence-associated gene profiles (VPs). Ten VPs were detected among wild boar isolates and 22 VPs among pig isolates, with only two VPs common to wild boars and pigs. The predominant VPs among isolates from wild boars (VP1, VP7) were different from those observed in pig isolates (VP16 and VP26). VP16 was detected exclusively in clinical pig isolates of serotype 9 and VP26 was detected in 71.4% of the serotype 2 clinical pig isolates. Further multilocus sequence typing (MLST) analysis showed a significant correlation association between certain VPs and STs (VP16 and VP17 with ST123 and ST125 and VP26 with ST1). In conclusion, the current study showed that combination of virulence-associated gene profiling and MLST analysis may provide more information of the relatedness of the S.suis strains from different animal species that could be useful for epidemiological purposes.
Examination of Australian Streptococcus suis isolates from clinically affected pigs in a global context and the genomic characterisation of ST1 as a predictor of virulence
Veterinary Microbiology, Volume 226, 2018, pp. 31-40
Streptococcus suis is a major zoonotic pathogen that causes severe disease in both humans and pigs. Australia’s pig herd has been quarantined for over 30 years, however S. suis remains a significant cause of disease. In this study, we investigated S. suis from 148 cases of clinical disease in pigs from 46 pig herds over a period of seven years, to determine the level of genetic difference from international isolates that may have arisen over the 30 years of separation. Isolates underwent whole genome sequencing, genome analysis and antimicrobial susceptibility testing. Data was compared at the core genome level to clinical isolates from overseas. Results demonstrated five predominant multi-locus sequence types and two major cps gene types (cps2 and 3). At the core genome level Australian isolates clustered predominantly within one large clade consisting of isolates from the UK, Canada and North America. A small proportion of Australian swine isolates (5%) were phylogenetically associated with south-east Asian and UK isolates, many of which were classified as causing systemic disease, and derived from cases of human and swine disease. Based on this dataset we provide a comprehensive outline of the current S. suis clones associated with disease in Australian pigs and their global context, with the main finding being that, despite three decades of separation, Australian S. suis are genomically similar to overseas strains. In addition, we show that ST1 clones carry a constellation of putative virulence genes not present in other Australian STs.
Species-specific real-time PCR assay for the detection of Streptococcus suis from clinical specimens
Diagnostic Microbiology and Infectious Disease, Volume 85, Issue 2, 2016, pp. 131-132
A real-time polymerase chain reaction was developed to detect all known strains of Streptococcus suis. The assay was highly specific, and sensitivity was <10 copies/assay for S. suis detection from clinical samples.
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