Development of novel Streptococcus equi vaccines with an assessment of their immunizing potentials and protective efficacies.

Strangles is a highly contagious disease of the equine upper respiratory tract caused by Streptococcus equi subspecies. Streptococcus equi subsp. equi (S. equi) and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was isolated, as local, hot, and field strains, from horses clinically suffering from respiratory distress. The isolated Streptococci were identified using bacteriological and molecular techniques. Four formulations of inactivated S. equi vaccines were developed and evaluated. The first formulation was prepared using the S. equi isolates, adjuvanted with MONTANIDE GEL adjuvant, while the second formulation was adjuvanted with MONTANIDE ISA-70 adjuvant. The other 2 formulations were inactivated combined vaccines prepared from both S. equi and S. zooepidemicus isolates. The 3rd formulation was the combined isolates adjuvanted with MONTANIDE GEL while the 4th formulation was the combined isolates adjuvanted with MONTANIDE ISA-70. The developed vaccines' physical properties, purity, sterility, safety, and potency were ensured. The immunizing efficacy was determined in isogenic BALB/c mice and white New Zealand rabbits using the passive hemagglutination test. Also, the antibodies' titer of the combined S. equi and S. zooepidemicus vaccine adjuvanted with MONTANIDE ISA-70 in foals was tracked using an indirect enzyme-linked immunosorbent assay. The protective efficacy of the developed vaccines was determined using a challenge test in both laboratory and field animal models, where a 75% protection rate was achieved. The combined vaccine proved to be more efficacious than the monovalent vaccine. Also, the MONTANIDE ISA-70 adjuvant provided significant protective efficacy than the MONTANIDE GEL. The current work is introducing a very promising mitigative and strategic controlling solution for strangles.

SzM protein of Streptococcus equi ssp. zooepidemicus triggers the release of neutrophil extracellular traps depending on GSDMD.

Streptococcus equissp.zooepidemicus (SEZ) is a crucial pathogen and contributes to various infections in numerous animal species. Swine streptococcicosis outbreak caused by SEZ has been reported in several countries in recent years. SzM protein is a cell membrane-anchored protein, which exhibits as an important virulence factor of SEZ. Effects of SzM protein on host innate immune need further study. Here, recombinant SzM (rSzM) protein of the SEZ was obtained, and mice were intraperitoneally injected with rSzM protein. We discovered that rSzM protein can recruit neutrophils into the injected site. In further study, neutrophils were isolated and treated with rSzM protein, NETs release were triggered by rSzM protein independently, and GSDMD protein was promoted-expressed and activated. In order to investigate the role of GSDMD in NETs formation, neutrophils isolated from WT mice and GSDMD-/- mice were treated with rSzM protein. The results showed that GSDMD deficiency suppressed the NETs release. In conclusion, SzM protein of SEZ can trigger the NETs release in a GSDMD-depending manner.

Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection.

Streptococcus equi subspecies equi is a pathogenic bacterium that causes strangles, a highly contagious respiratory infection in horses and other equines. The limitations of current vaccines against S. equi infection warrants the development of an affordable, safe, and effective vaccine. Because gram-positive extracellular vesicles (EVs) transport various immunogenic antigens, they are attractive vaccine candidates. Here, we purified the EVs of S. equi ATCC 39506 and evaluated them as a vaccine candidate against S. equi infection in mice. As an initial step, comparative proteomic analysis was performed to characterize the functional features of the EVs. Reverse vaccinology and knowledge-based annotations were then used to screen potential vaccine candidates (PVCs) for S. equi ATCC 39506. Finally, 32 PVCs were found to be enriched in the EV fraction, suggesting the usefulness of this fraction as a vaccine. Importantly, a significantly higher survival rate after S. equi infection was detected in mice immunized with S. equi-derived EVs via the intraperitoneal route than in mice immunized with heat-killed bacteria. Of note, immunoprecipitation-mass spectrometry results validated various immunogenic antigens within the EV proteome. In conclusion, our results suggest that S. equi-derived EVs can serve as a vaccine candidate against S. equi infection.

Effects of srtA variation on phagocytosis resistance and immune response of Streptococcus equi.

Strangles, which is caused by Streptococcus equi subspecies equi (S. equi), is one of the most prevalent equine infectious diseases with worldwide distribution and leads to serious economic loss in the horse industry. Sortase A (srtA) is a transpeptidase that anchors multiple virulence-associated surface proteins to the cell surface of S. equi. srtA plays a major role in S. equi infection and colonization of the host cell. In this study, we aimed to investigate the effects of srtA mutation on the phagocytic activity and immunogenicity of S. equi. The point-mutated recombinant sortases, including srtA-HT1112 (I88V), srtA-5012 (R147G), and srtA-ZZM17 (control), were expressed, purified, and used to immunize the mouse models. Phagocytic activity was assessed using equine polymorphonuclear cells, whereas opsonophagocytic function and adherence inhibition were measured using the antiserum of these mutants. Mouse serum antibody, bacterial load, and weight gain were also measured. The srtA-HT1112 (I88V) mutant showed significantly enhanced antiphagocytic capability, and its antiserum exhibited increased adherence inhibition activity. In addition, the srtA-HT1112 (I88V) mutant presented the highest lung bacterial load and lowest protection rate (50%) after the challenge with S. equi ZZM17. The srtA-5012 (R147G) mutant exhibited a high IgG2a level and protection rate (62.5%-75%) and the lowest lung bacterial load. These results indicate that the I88V mutation is associated with a high antiphagocytic activity, whereas R147G mutation is associated with the decreased lung bacterial load. Our findings may be useful for the evaluation and development of vaccines.

Vaccination of yearling horses against poly-N-acetyl glucosamine fails to protect against infection with Streptococcus equi subspecies equi.

Strangles is a common disease of horses with worldwide distribution caused by the bacterium Streptococcus equi subspecies equi (SEE). Although vaccines against strangles are available commercially, these products have limitations in safety and efficacy. The microbial surface antigen ß 1→6 poly-N-acetylglucosamine (PNAG) is expressed by SEE. Here we show that intramuscular (IM) injection alone or a combination of IM plus intranasal (IN) immunization generated antibodies to PNAG that functioned to deposit complement and mediate opsonophagocytic killing of SEE ex vivo. However, immunization strategies targeting PNAG either by either IM only injection or a combination of IM and IN immunizations failed to protect yearling horses against infection following contact with infected horses in an experimental setting. We speculate that a protective vaccine against strangles will require additional components, such as those targeting SEE enzymes that degrade or inactivate equine IgG.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

Novel Streptococcus equi strains causing strangles outbreaks in Arabian horses in Egypt.

Strangles displays a major challenge to veterinary medicine worldwide. However, no data on Streptococcus equi subsp. equi (S. equi) M protein alleles have been reported so far from Arabian horses. We report here for the first time the S. equi SeM alleles causing strangles in Arabian horses, and the associated risk factors for the disease. Duplicate samples from one hundred Arabian horses with acute strangles in confirmed outbreaks and sporadic cases were analysed by phenotypic methods and multiplex polymerase chain reaction (PCR) targeting streptokinase precursor, seeI and sodA genes. PCR and sequencing of S. equi SeM gene were employed for strains typing, and the four superantigens were determined among the allelic variants. Direct-sample PCR confirmed and highly positively correlated (r = .85) with the phenotypic results, and detected S. equi in five samples more than the conventional culture. A combination of multiplex PCR from samples and culture could successfully identify S. equi (92%), S. zooepidemicus (5%) and S. equisimilis (3%). SeM typing demonstrated five SeM alleles, including four previously unidentified alleles that were deposited in the PubMLST-SeM database. SeM-139 and SeM-141 are related to some strains that were recently recovered from donkeys in China. SeM-140 and SeM-199 are related to a group of alleles from horses in Europe. Variation in the presence of seeM, seeH and seeL superantigens was found across the four novel alleles without interference with the severity of strangles and clinical presentation seen in different outbreaks. Horse age was the most important factor in developing strangles, followed by seasonality and the diagnosis of strangles in the previous year. These new findings comprise a significant contribution to the horse industry through the identification of novel S. equi SeM types that may bolster measures for strangles control as the identified SeM alleles will certainly help in the development of SeM-containing vaccine.

The feto-maternal immune response to equine placentitis.

PROBLEM: Ascending placentitis is one of the leading causes of abortion in the horse. Minimal work has focused on its effect on fetal fluids or the antenatal immune response of the fetus. METHODOLOGY: Placentitis was induced via transcervical inoculation of Streptococcus equi ssp Zooepidemicus, and fluids/serum/tissues were collected 4-6 days later following euthanasia. Cytokine concentrations were detected using a multiplex immunoassay within fetal fluids (amniotic and allantoic) and serum (maternal and fetal) in inoculated and control mares. In addition, tissues from fetal (spleen, liver, lung, umbilicus, amnioallantois) and maternal (spleen, liver, lung, chorioallantois, endometrium) origin were analyzed in inoculated and control mares utilizing qPCR for expression of cytokines. RESULTS: No difference in cytokine concentrations in maternal or fetal serum was noted between inoculated and control mares. Concentrations of IL-1ß, IL-6, IL-10, and GRO were upregulated in the amniotic fluid following inoculation, with a trend toward higher IL-6 concentration in allantoic fluid. The amnioallantoic tissue separating the two fluids had higher expression of IL-1ß and IL-6 following inoculation, while chorioallantois and endometrium upregulated IL-1ß and IL-8 expression. IL-1ß was upregulated in the maternal spleen following inoculation. Fetal spleens were upregulated in expression of IL-1ß, GRO, and IL-6, while IL-6 was higher in fetal liver after inoculation than in controls. CONCLUSION: The maternal response to placentitis is primarily pro-inflammatory while the fetus appears to play a regulatory role in this inflammation. Additionally, amniotic fluid sampling may be more diagnostic of ascending placentitis than circulating cytokines.

Antibody kinetics and immune profile analysis of a Streptococcus equi DNA vaccine expressing the FljB and SeM fusion protein in murine and equine models.

Strangles is a highly prevalent, extremely contagious, and occasionally lethal infectious disease affecting horses worldwide. Prophylactic antibiotics are ineffective in prevention of disease but are recommended for exposed horses at the first sign of fever and any horse obviously ill from strangles or with complications and there is an urgent need of a cost-effective, safe, efficacious vaccine. In the present study, we sought to develop effective vaccines by fusing the Streptococcus equi subspecies equi (S. equi) antigen SeM with the flagellin of Salmonella abortus equi FljB. We also explored the immunogenicity and efficacy of this candidate vaccine in mice and horses by intramuscular injection. Mice and horses immunized with FljB-SeM DNA vaccine showed high levels of specific antibody and increased production of IFN-γ and IL-4. This confirmed that both Th1 and Th2 type responses were induced. The mice survival rate was significantly higher after immunization with FljB-SeM than with SeM alone. The FljB-SeM DNA could strengthen both the Th1 and Th2 immune responses compared to SeM and could provide better protection against S. equi. This technique could help develop a candidate vaccine for S. equi infection.

Identification of a surface protective antigen, MAP of Streptococcus equi subspecies zooepidemicus.

Streptococcus equi subspecies zooepidemicus (SEZ) is a zoonotic pathogen with adhesive and invasive properties. Due to the shortcomings of antibiotics and traditional inactivated vaccine, identifying protective antigens against SEZ would be helpful to the development of novel vaccines. MAP has been identified as a membrane anchored protein with a typical LPXTG-like cell wall-anchored motif. In present study, the objective was to evaluate the effects of MAP as a subunit vaccine with mouse model. The Western blot analysis shown that the purified recombinant MAP presented good immunoreactive to convalescent porcine sera against SEZ. The protein could elicit a remarkable humoral antibody response and protect 80% of mice against lethal dose challenge of SEZ in mouse model. Moreover, the hyperimmune sera against MAP could efficiently kill the bacteria in whole blood killing assay and conferred significant protection against SEZ in passive immunization experiments. This study suggests with good reasons that MAP could be a novel and effective vaccine candidate for SEZ.

Chemical Modification of the N-Acetyl Moieties of Hyaluronic Acid from Streptococcus equi for Studies in Cytokine Production.

Partial N-deacetylation and certain N-reacylations of low-molecular-weight hyaluronic acid (hyaluronan) abate its proinflammatory properties in mammalian systems. Here, we describe the treatment of bacterial hyaluronic acid by hydrazine or NaOH to yield smaller partially deacetylated polymers. These N-deacetylated polymers can be reacylated with acyl anhydrides to yield substituted hyaluronic acid derivatives of equivalent size and equimolar N-acyl substitutions.

Strangles, convalescent Streptococcus equi subspecies equi M antibody titers, and presence of complications.

BACKGROUND: Streptococcus equi subspecies equi infection elicits M protein antibody titers in equids. Interpretation of titers is not generally accepted. HYPOTHESIS: The magnitude of S. equi M protein (SeM) antibody titer after infection (titer ≥1:12 800) will be useful to monitor for the presence of complications or the risk of development of complications. ANIMALS: Forty-eight horses on 1 farm involved in strangles outbreak. METHODS: Clinical and observational study. S. equi M protein antibody titers were measured on all horses 8 weeks after infection and select horses 12 and 28 weeks after infection. Horses were categorized: no disease, uncomplicated case, persistent guttural pouch (GP) infection, or complicated cases (metastatic abscesses, purpura hemorrhagica, secondary infections, and dysphagia). Category was compared to titer. RESULTS: Twenty-eight of 48 (58%) developed clinical signs of S. equi infection. Of those, 11 (39%) had uncomplicated strangles, 9 (21%) had persistent GP infection, 5 (18%) were complicated cases, and 3 (11%) had both persistent GP infection and complications. Thirty-three percent of horses (16 of 48) had SeM antibody titers ≥1:12 800 eight weeks after infection. Of horses with titers ≥1:12 800, 6 of 16 had evidence of complications. Of complicated cases, 6 of 8 had titers ≥1:12 800. In this outbreak, the sensitivity (75%; 95% CI [confidence interval] 45-105) for a SeM antibody titer ≥1:12 800 detecting complications was higher than the specificity (43%; 95% CI 23-64). CONCLUSIONS AND CLINICAL IMPORTANCE: This outbreak demonstrates that SeM antibody titers can be increased after infection (≥1:12 800) in the absence of complications of strangles.

Characterization of SeseC_01411 as a surface protective antigen of Streptococcus equi ssp. zooepidemicus.

Streptococcus equi ssp. zooepidemicus (Streptococcus zooepidemicus, SEZ) is a commensal bacterium related to opportunistic infections of many species, including humans, dogs, cats, and pigs. SeseC_01411 has been proven to be immunogenic. However, its protective efficacy remained to be evaluated. In the present study, the purified recombinant SeseC_01411 could elicit a strong humoral antibody response and protect against lethal challenge with virulent SEZ in mice. Our finding confirmed that SeseC_01411 distributes on the surface of SEZ. In addition, the hyperimmune sera against SeseC_01411 could efficiently kill the bacteria in the phagocytosis test. The present study identified the immunogenic protein, SeseC_01411, as a novel surface protective antigen of SEZ.

Phagocytosis, bacterial killing, and cytokine activation of circulating blood neutrophils in horses with severe equine asthma and control horses.

OBJECTIVE To evaluate in vitro phagocytosis and bactericidal activity of circulating blood neutrophils in horses with severe equine asthma and control horses and to determine whether circulating blood neutrophils in horses with severe equine asthma have an increase in expression of the proinflammatory cytokine tumor necrosis factor (TNF)-α and the chemokine interleukin (IL)-8 and a decrease in expression of the anti-inflammatory cytokine IL-10 in response to bacteria. ANIMALS 6 horses with severe equine asthma and 6 control horses. PROCEDURES Circulating blood neutrophils were isolated from horses with severe equine asthma and control horses. Phagocytosis was evaluated by use of flow cytometry. Bactericidal activity of circulating blood neutrophils was assessed by use of Streptococcus equi and Streptococcus zooepidemicus as targets, whereas the cytokine mRNA response was assessed by use of a quantitative PCR assay. RESULTS Circulating blood neutrophils from horses with severe equine asthma had significantly lower bactericidal activity toward S zooepidemicus but not toward S equi, compared with results for control horses. Phagocytosis and mRNA expression of TNF-α, IL-8, and IL-10 were not different between groups. CONCLUSIONS AND CLINCAL RELEVANCE Impairment of bactericidal activity of circulating blood neutrophils in horses with severe equine asthma could contribute to an increased susceptibility to infections.

Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles-Revised Consensus Statement.

This consensus statement update reflects our current published knowledge and opinion about clinical signs, pathogenesis, epidemiology, treatment, complications, and control of strangles. This updated statement emphasizes varying presentations in the context of existing underlying immunity and carrier states of strangles in the transmission of disease. The statement redefines the "gold standard" for detection of possible infection and reviews the new technologies available in polymerase chain reaction diagnosis and serology and their use in outbreak control and prevention. We reiterate the importance of judicious use of antibiotics in horses with strangles. This updated consensus statement reviews current vaccine technology and the importance of linking vaccination with currently advocated disease control and prevention programs to facilitate the eradication of endemic infections while safely maintaining herd immunity. Differentiation between immune responses to primary and repeated exposure of subclinically infected animals and responses induced by vaccination is also addressed.

Strangvac: A recombinant fusion protein vaccine that protects against strangles, caused by Streptococcus equi.

The host-restricted pathogen Streptococcus equi causes strangles in the horse, which is characterised by abscessation of the lymph nodes of the head and neck. The disease is endemic throughout the world causing considerable welfare and economic cost to the horse industry. Here we report the results of three studies where ponies were vaccinated with combinations of recombinant fusion proteins to optimise vaccine production and the level of protection conferred. Optimal protection was conferred by a prototype multicomponent subunit vaccine, Strangvac 4, which contained eight proteins CNE, SclC, SclF, SclI, EAG (fused as CCE), SEQ_402, SEQ_0256 (fused as Eq85) and IdeE. Across the three experiments only three of 16 ponies vaccinated with Strangvac 4 became pyretic compared to all 16 placebo-vaccinated control ponies (P < .001). S. equi was recovered from the lymph nodes of eight Strangvac 4-vaccinated and 15 control ponies (P = .016). None of the ponies vaccinated with Strangvac 4, or the other prototype vaccines developed adverse reactions following vaccination. Our data provide evidence in support of the further clinical development of the Strangvac 4 vaccine.

Decreased Clinical Severity of Strangles in Weanlings Associated with Restricted Seroconversion to Optimized Streptococcus equi ssp equi Assays.

BACKGROUND: Streptococcus equi ssp. equi causes characteristic clinical signs that are most severe in young horses, including fever, purulent nasal discharge, and lymph node abscessation in the head region. HYPOTHESIS/OBJECTIVES: Clinical, serologic, and microbiologic factors related to unexpectedly mild disease severity in a natural outbreak of strangles in immunologically naïve weanlings were investigated. ANIMALS: One-hundred and twelve warmblood weanlings. METHODS: Prospective longitudinal observational study of a natural outbreak of strangles. The entire cohort was examined at the peak of the outbreak by deep nasal swabs for culture and quantitative PCR (qPCR) for the presence of S. equi and clinically and serologically in a sequential manner by an optimized ELISA from the index case throughout the outbreak until resolution. Descriptive statistics were calculated and comparisons made using a nondirectional Wilcoxon signed-rank test. RESULTS: Outbreak morbidity was 53%, with 9 of 14 horses culture positive and 26 of 53 horses qPCR positive for S. equi lacking clinical signs characteristic of strangles. By resolution, 91 of 112 had seroconverted to Antigen A by ELISA but seroconversion to antigen C (part of the SeM protein) was minimal. Sequencing of the isolates detected no alterations in the SeM protein, but identified a 61 bp deletion in the gene SEQ_0402. CONCLUSIONS AND CLINICAL IMPORTANCE: Absence of clinical signs alone in naïve horses may be an insufficient criterion to release horses from strangles quarantine measures. Restricted seroconversion to antigen C may have been associated with decreased clinical severity. The role of a minor gene deletion in SEQ_0402 in the virulence of S. equi warrants further investigation.

Identification and characterization of a novel protective antigen, Sec_205 of Streptococcus equi ssp. Zooepidemicus.

Streptococcus equi ssp. zooepidemicus (SEZ) is an important pathogen of swine streptococcal diseases and can infect a wide range of animals as well as human beings. The absence of effective vaccine confounds the control of SEZ infection. Sec_205, a novel protein identified in the previous study, was inducibly over-expressed in Escherichia coli in the present study. The purified recombinant protein could elicit a significant humoral antibody response and provide efficient protection against lethal challenge of SEZ C55138 in mouse model. The protection against SEZ infection was mediated by specific antibodies to Sec_205 to some extent and was identified by the passive protection assay. The Sec_205 was an in vivo-induced antigen confirmed by the real-time PCR and could adhere to the Hep-2 cells by the inhibition assay. These suggest that Sec_205 may play a vital role in pathogenicity and serve as a new vaccine candidate against SEZ infection.

A case-control study developing a model for predicting risk factors for high SeM-specific antibody titers after natural outbreaks of Streptococcus equi subsp equi infection in horses.

OBJECTIVE To develop a risk prediction model for factors associated with an SeM-specific antibody titer ≥ 3,200 in horses after naturally occurring outbreaks of Streptococcus equi subsp equi infection and to validate this model. DESIGN Case-control study. ANIMALS 245 horses: 57 horses involved in strangles outbreaks (case horses) and 188 healthy horses (control horses). PROCEDURES Serum samples were obtained from the 57 cases over a 27.5-month period after the start of outbreaks; serum samples were obtained once from the 188 controls. A Bayesian mixed-effects logistic regression model was used to assess potential risk factors associated with an antibody titer ≥ 3,200 in the case horses. A cutoff probability for an SeM-specific titer ≥ 3,200 was determined, and the model was externally validated in the control horses. Only variables with a 95% credibility interval that did not overlap with a value of 1 were considered significant. RESULTS 9 of 57 (6%) case horses had at least 1 titer ≥ 3,200, and 7 of 188 (3.7%) of control horses had a titer ≥ 3,200. The following variables were found to be significantly associated with a titer ≥ 3,200 in cases: farm size > 20 horses (OR, 0.11), history of clinically evident disease (OR, 7.92), and male sex (OR, 0.11). The model had 100% sensitivity but only 24% specificity when applied to the 188 control horses (area under the receiver operating characteristic curve = 0.62.) CONCLUSIONS AND CLINICAL RELEVANCE Although the Bayesian mixed-effects logistic regression model developed in this study did not perform well, it may prove useful as an initial screening tool prior to vaccination. We suggest that SeM-specific antibody titer be measured prior to vaccination when our model predicts a titer ≥ 3,200.

Zoonotic necrotizing myositis caused by Streptococcus equi subsp. zooepidemicus in a farmer.

BACKGROUND: Streptococcus equi subsp. zooepidemicus is a beta-hemolytic group C streptococcus mainly causing infections in domesticated animals. Here we describe the first case of zoonotic necrotizing myositis caused by this bacterium. CASE PRESENTATION: The patient was a 73-year-old, previously healthy farmer with two asymptomatic Shetland ponies in his stable. After close contact with the ponies while feeding them, he rapidly developed erythema of his left thigh and sepsis with multiple organ failure. The clinical course was severe and complicated, requiring repetitive surgical excision of necrotic muscle, treatment with vasopressors, mechanical ventilation and continuous venovenous hemofiltration, along with adjunctive hyperbaric oxygen therapy. The patient was discharged from hospital at day 30, without obvious sequelae. The streptococcal isolate was identified as Streptococcus equi by MALDI-ToF MS, and was later assigned subspecies identification as S. equi subsp. zooepidemicus. Multilocus sequence typing identified the strain as a novel sequence type (ST 364), closely related to types previously identified in horses and cattle. A focused proteomic analysis revealed that the ST 364 expressed putative virulence factors similar to that of Streptococcus pyogenes, including homologues of the M protein, streptodornases, interleukin 8-protease and proteins involved in the biosynthesis of streptolysin S. CONCLUSION: This case illustrates the zoonotic potential of S. equi subsp. zooepidemicus and the importance of early clinical recognition, rapid and radical surgical therapy, appropriate antibiotics and adequate supportive measures when necrotizing soft tissue infection is suspected. The expression of Streptococcus pyogenes-like putative virulence determinants in ST 364 might partially explain the fulminant clinical picture.