Streptococcus Equi Ssp. Zooepidemicus Research Articles

Neutrophil accumulation raises defence against Streptococcus equi ssp. zooepidemicus in the absence of Gasdermin D

Streptococcus equi ssp. zooepidemicus (SEZ) predominantly acts as a zoonotic pathogen, capable of infecting a diverse range of animal species including human. Gasdermin D (GSDMD) exhibited comprehensive functions in host against different pathogenic microorganism. This study aimed to investigate the role of GSDMD in host against SEZ. Mice were administrated with SEZ via intranasal intubation for 24 h (3 × 106CFU), GSDMD protein expression significantly increased in the lung tissue of mice infected with SEZ. For further research on the role of GSDMD during SEZ infection, GSDMD-/- mice and WT mice were treated with SEZ via intranasal intubation for 24 h (3 × 106CFU). GSDMD-/- mice showed less severe lung tissue due to fewer bacteria colonization. Numerous neutrophils were recruited into lung tissues in GSDMD-/- mice, related to the release of CXCL1 and CXCL2 regulated by p65 phosphorylation. In further study, neutrophils of WT and GSDMD-/- mice were isolated and treated with SEZ (multiplicity of infection, MOI = 10, 4 h). The absence of GSDMD alleviated the death of neutrophils, in addition, GSDMD deficiency could promote translocation of p65 from the cytoplasm into the nucleus in neutrophil, which may contribute to the release of IL-1β and TNF-α. This study demonstrated a novel function of GSDMD in host immune response to SEZ invading, indicating that GSDMD deficiency ameliorated SEZ infection through enhancing neutrophil accumulation into infected site, and activating NF-κB pathway in neutrophil to release cytokines against SEZ. Our study suggested that inhibition of host GSDMD may be an effective method against SEZ.

Isolation and characterization of bacteriophages specific to selected equine genital pathogens

Chronic bacterial endometritis is among the major problems in equine reproduction. Streptococcus equi ssp. zooepidemicus (SEZ), Klebsiella pneumoniae (KP), and Pseudomonas aeruginosa (PA) are among the most important pathogens for the condition. Bacteriophages (phages) are the viruses of bacteria, having a species- and strain-specific lytic activity. Due to their bactericidal capacities, phages can be used as an alternative treatment approach for bacterial infections. The study aimed to isolate and characterize phages with lytic activity against Streptococcus equi ssp. zooepidemicus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phage samples were obtained from manure, uterine lavage fluid and drain water of horse husbandries (n=14). For isolation of phages, bacterial isolates of the equine genital tract (SEZ (n=1), KP (n=1), and PA (n=1)) were inoculated with the phage samples. For purification, bacteriophage plaques were repeatedly cultivated, harvested and filtered. For determining the host-range, the obtained phages were inoculated with bacterial isolates (SEZ (n=37), KP (n=26), and PA (n=33)). In total, 28 bacteriophages were isolated (against SEZ (n=13), KP (n=6), and PA (n=9)). Bacteriolysis was achieved in 32/37 Streptococcus-, 16/26 Klebsiella-, and 14/33 Pseudomonas-isolates. The bacteriophage showing the broadest host range covered 81% of all tested Streptococcus-isolates (30/37). Host ranges of Klebsiella pneumoniae- (9/26) and Pseudomonas aeruginosa-specific bacteriophages (14/33) covered a lower proportion of tested isolates. Seven bacteriophages (SEZ (n=3), KP (n=3), PA (n=1)) were selected according to theirhost ranges and submitted to electron microscopy for further characterization. Electron micrographs revealed that the selected bacteriophages belonged either to the family Demerecviridae (n=1), Myoviridae (n=1), Podoviridae (n=3) or Siphoviridae (n=2), which was confirmed by sequencing. Next, planktonic killing assay was conducted, incubating liquid cultures of Streptococcus equi ssp. zooepidemicus-, Klebsiella pneumoniae- and Pseudomonas aeruginosa-isolates in presence and absence of a specific bacteriophage and monitoring growth by optical density measurements for 24h. Significantly decreased bacterial growth, as determined by comparing the area under the curve (AUC), was observed in presence of 0/3 Streptococcus equi ssp. zooepidemicus-, 1/1 Pseudomonas aeruginosa- and 3/3 Klebsiella pneumoniae-specific phages, using a multiplicity of infection of 1 (MOI1; one phage per host cell). At MOI10 the most effective Streptococcus-phage reduced the AUC by 61%; the most effective Klebsiella-phage resulted in 95% reduction using MOI1 and for the Pseudomonas-phage AUC was reduced by 14% at MOI100. In conclusion, isolation of phages specific to equine uterine pathogens resulted in 28 purified phages. Seven phages were selected and further characterized by efficacy testing, sequencing and electron microscopy. Results of the in vitro host range determination and planktonic killing assay are promising, suggesting further investigations and the development of an alternative treatment approach for uterine infections in mares.

Identification of a novel protective antigen, 3-oxoacyl-[acyl-carrier-protein] synthase II of Streptococcus equi ssp. zooepidemicus which confers protective effects

Streptococcus equi ssp. zooepidemicus (SEZ) is an important swine pathogen and responsible for a wide variety of infections in many animal species. FabF was a novel protein identified in the previous study. However, its protective efficacy remained to be evaluated. In this study, recombinant fabF of SEZ was expressed and showed a strong immunoreactivity with mini-pig convalescent sera. Study in mice revealed that the recombinant protein induced a marked antibody response and protected 80% of mice against SEZ infection. The hyperimmune sera against fabF could efficiently kill the bacteria in the phagocytosis test. In addition, it was also found that anti- fabF antibodies can significantly inhibit the formation of SEZ biofilm. These study suggest that fabF may represent immunogens of interest for vaccine development against SEZ infection.

Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares

Anatomical and molecular changes in the cervical barrier in women are a fundamental part of the pathogenesis of pregnancy loss associated with chorioamnionitis. However, there is little information regarding changes in the cervix associated with ascending infection in pregnant mares. To better characterize morphological and molecular changes in the cervix during placentitis, we examined full thickness histology and mRNA expression for a number of inflammatory and endocrine factors in the mucosa and stroma of the cervix of mares (n = 5) after experimental induction of placentitis via transcervical inoculation with Streptococcus equi ssp zooepidemicus at approximately 290d of gestation. Gestationally age-matched mares (n = 4) served as controls. Target transcripts included steroid receptors (PGR, ESR1 and 2), OXTR, prostaglandins synthases and receptors (PTGS1, PTGS2, PGES, PGFS, PTGER2 and PTGER4), cytokines (IL1b, IL6, CLCX8, IL10 and TNFα) and acute phase proteins (SAA). Histologically, a marked modification in the cervical epithelia and stroma was characterizing cervicitis. Additionally, the mRNA expression of IL1β, IL6, CXCL8, SAA and PTGS2 was greater (P < 0.05) in both mucosa and stroma of the inoculated mares; whereas TNFα, IL10 and PGES were upregulated (P < 0.05) only in the cervical mucosa. Progesterone receptor, ESR1 and PTGER4 were upregulated in the cervical stroma of placentitis mares. In conclusion, the cervical response to placentitis was characterized by an upregulation of inflammatory cytokines that was accompanied by induction of PTGS2 and PGES. Further, receptors known to be associated with relaxation of the cervix in other species (ESR1 and PTGER4) were upregulated in the cervical stroma of placentitis mares. These findings indicate that the cervix is not only a physical barrier but that it has an active role in the pathogenesis of ascending placentitis.

Evaluating the efficacy of an attenuated Streptococcus equi ssp. zooepidemicus vaccine produced by multi-gene deletion in pathogenicity island SeseCisland_4

Streptococcus equi ssp. zooepidemicus (SEZ) is a pathogen associated with a wild range of animal species. Frequent outbreaks have occurred in recent years in pigs, horses, goats and dogs which is liable to infect humans. There is a lack of efficient vaccines against this disease and the occurrence of antibiotic resistance may render drug therapies ineffective. In this study, gene deletion mutant (ΔSEZ) in pathogenicity islands SeseCisland_4 was constructed. The mutant ΔSEZ had a 52-fold decrease in 50% lethal dose (LD50) and had less capacity to adhere epithelial cells. Importantly, immunization of mice with attenuated vaccine ΔSEZ at the dose of 102 colony-forming units (CFU) mL−1 elicited a significant humoral antibody response, with an antibody titer of 1:12 800. Therefore, 102 CFU mL−1 might be used as the appropriate immune dose for the attenuated vaccine ΔSEZ, which provided mice with efficient protection against virulent SEZ. In addition, the hyperimmune sera against 102 CFU mL−1 attenuated vaccine ΔSEZ could confer significant protection against virulent SEZ infection in the passive immunization experiment and exhibited efficient bactericidal activity in the whole blood assay. Meanwhile, no viable bacteria was detected in blood when mice were immunized with ΔSEZ at the dose of 102 CFU mL−1via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 102 CFU mL−1 could confer significant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.

Equine placentitis is associated with a downregulation in myometrial progestin signaling†.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n=6) with uninoculated gestationally matched mares as controls (n=4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P<0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P<0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1β and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation.

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.

Virulent and Vaccine Strains of Streptococcus equi ssp. zooepidemicus Have Different Influences on Phagocytosis and Cytokine Secretion of Macrophages

Swine streptococcosis is a significant threat to the Chinese pig industry, and Streptococcus equi ssp. zooepidemicus (SEZ) is one of the major pathogens. SEZ ATCC35246 is a classical virulent strain, while SEZ ST171 is a Chinese attenuated vaccine strain. In this study, we employed stable isotope labeling by amino acids in cell culture and liquid chromatography-mass spectrometry (LC-MS) to determine the differential response of macrophages to infection by these two strains. Eighty-seven upregulated proteins and 135 downregulated proteins were identified. The proteomic results were verified by real-time polymerase chain reaction for 10 chosen genes and Western blotting for three proteins. All differentially abundant proteins were analyzed for their Gene Ontology and Kyoto Encyclopedia of Genes and Genomes annotations. Certain downregulated proteins were associated with immunity functions, and the upregulated proteins were related to cytomembrane and cytoskeleton regulation. The phagocytosis rate and cytokine genes transcription in Raw264.7 cells during SEZ ATCC35246 and ST171 infection were detected to confirm the bioinformatics results. These results showed that different effects on macrophage phagocytosis and cytokine expression might explain the different phenotypes of SEZ ATCC35246 and ST171 infection. This research provided clues to the mechanisms of host immunity responses to SEZ ST171and SEZ ATCC35246, which could identify potential therapy and vaccine development targets.

A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation.

The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis.

SILAC and LC-MS/MS identification of Streptococcus equi ssp. zooepidemicus proteins that contribute to mouse brain microvascular endothelial cell infection.

Streptococcus equi ssp. zooepidemicus (SEZ) causes meningitis in both humans and animals. Some dissociative proteins of SEZ are cytotoxic to mouse brain microvascular endothelial cells (mBMECs) and may contribute to the penetration of SEZ across the blood-brain barrier (BBB). In this study, the ability of SEZ to penetrate across an in vitro BBB model was confirmed. We used stable isotope labeling with amino acids in cell culture (SILAC) to label SEZ proteins with heavy or light isotope-tagged amino acids, along with LC-MS/MS to determine which SEZ proteins were involved in interactions with mBMECs. The efficiency of SEZ protein isotope labeling was 94.7%, which was sufficient for further analysis. Forty-nine labeled peptides were identified as binding to mBMECs, which matched to 25 SEZ proteins. Bioinformatic analysis indicated that most of these proteins were cytoplasmic. These proteins may have functions in breaching the host BBB, and some of them are known virulence factors in other bacteria. Indirect immunofluorescence results indicated that SEZ enolase had binding activity toward mBMECs. Protective test results showed that enolase was a protective antigen against SEZ infection. This research is the first application of SILAC combined with LC-MS/MS to identify SEZ proteins that may contribute to the infection of mBMECs and potentially show functions related to breaching the BBB. The outcomes provide many future avenues for research into the mechanism of SEZ-induced meningitis.

Biofilm Formation of Streptococcus equi ssp. zooepidemicus and Comparative Proteomic Analysis of Biofilm and Planktonic Cells

Streptococcus equi ssp. zooepidemicus (SEZ) is responsible for a wide variety of infections in many species, including pigs, horses and humans. Biofilm formation is essential for pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. However, the ability of SEZ to form biofilms is unclear. Furthermore, the mechanisms underlying SEZ biofilm formation and their attributes are poorly understood. In this study, scanning electron microscopy (SEM) demonstrated that SEZ strain ATCC35246 formed biofilms comprising a thick, heterogeneous layer with clumps on the coverslips when incubated for 24 h. In addition, we used a two-dimensional gel electrophoresis (2-DE) based approach to characterize differentially expressed protein in SEZ biofilms compared with their planktonic counterparts. The results revealed the existence of 24 protein spots of varying intensities, 13 of which were upregulated and 11 were downregulated in the SEZ biofilm compared with the planktonic controls. Most of proteins expressed during biofilm formation were associated with metabolism, adhesion, and stress conditions. These observations contribute to our understanding of the SEZ biofilm lifestyle, which may lead to more effective measures to control persistent SEZ infections.

A novel vaccine against Porcine circovirus type 2 (PCV2) and Streptococcus equi ssp. zooepidemicus (SEZ) co-infection

To develop a vaccine against Porcine circovirus type 2 (PCV2) and Streptococcus equi ssp. zooepidemicus (SEZ) co-infection, the genes of porcine IL-18, capsid protein (Cap) of PCV2 and M-like protein (SzP) of SEZ were inserted into the swinepox virus (SPV) genome by homologous recombination. The recombinant swinepox virus rSPV-ICS was verified by PCR and indirect immunofluorescence assays. To evaluate the immunogenicity of rSPV-ICS, 28 PCV2 and SEZ seronegative Bama minipigs were immunized with rSPV-ICS (n=8), commercial PCV2 vaccine and SEZ vaccine (n=8) or wild type SPV (n=8). The results showed that SzP-specific antibody and PCV2 neutralizing antibody of the rSPV-ICS immunized group increased significantly compared to the wild type SPV treated group after vaccination and increased continuously over time. The levels of IL-4 and IFN-γ in the rSPV-ICS immunized group were significantly higher than the other three groups, respectively. After been co-challenged with PCV2 and SEZ, 87.5% piglets in rSPV-ICS immunized group were survived. Significant reductions in gross lung lesion score, histopathological lung lesion score, and lymph node lesion score were noticed in the rSPV-ICS immunized group compared with the wtSPV treated group. The results suggested that the recombinant rSPV-ICS provided piglets with significant protection against PCV2-SEZ co-infection; thus, it offers proof-of-principle for the development of a vaccine for the prevention of these swine diseases.

Identification of genes transcribed by Streptococcus equi ssp. zooepidemicus in infected porcine lung

Streptococcus equi ssp. zooepidemicus (SEZ) is a Gram-positive bacterium responsible for respiratory tract infection, septicemia, meningitis, endocarditis and arthritis in swine and humans. However, the expression and regulation of SEZ genes during an infection in vivo are poorly understood. In this study, we focused on the identification of SEZ genes preferentially expressed in vivo during infection in pigs. This study identified 45 SEZ genes that were upregulated in infected porcine lung tissues using the selective capture of transcribed sequences (SCOTS) technique and comparative dot-blot analysis, followed by quantitative RT-PCR validation. The identified genes were characterized into 6 functional categories: metabolism, cell wall-associated, stress response, transporters, regulators and unknown functions. Our study successfully identified multiple genes, which can deepen our understanding about SEZ pathogenesis and infer probable virulence factors. It will promote the development of novel vaccines and therapies about this pathogen for further study.

Identification and Characterization of Putative Virulent Genes in Streptococcus equi ssp. zooepidemicus

Suppression subtractive hybridization (SSH) was performed with virulent strain ATCC35246 and avirulent strain ST171 to identify novel genes associated with virulence in Streptococcus equi ssp. zooepidemicus (SEZ). There were fourteen genomic regions that only presented in virulent strain ATCC35246. These regions encoded 14 proteins, some of them were homologous to proteins associated with cellular surface structure, molecular synthesis, energy metabolism, regulation, transport systems, and other unknown functions. Primers for 6 particular regions were designed from the already published SEZ sequence. Then, we used PCR to evaluate the distribution and conservation of these 6 DNA fragments in various SEZ strains collected from different sources, regions, groups, and times. The results showed that these 6 DNA fragments were widely distributed in SEZ strains, yet they were not existence in the avirulent strain ST171. Moreover, these fragments could not be detected in other Streptococcus groups.

The capsule of Streptococcus equi ssp. zooepidemicus is a target for attenuation in vaccine development

Streptococcus equi ssp. zooepidemicus (SEZ) is an important pathogen associated with a wide range of diseases in many mammalian species. The development of novel effective vaccines would be beneficial to control SEZ infection. In the present study, the importance of the SEZ capsule was examined using a newly constructed capsule-deficient mutant ΔhasB strain. Transmission electron microscopy confirmed a decrease in the abundance of extracellular capsular polysaccharide on the mutant SEZ. Compared to the parental wild-type SEZ, the ΔhasB mutant was highly attenuated in mice and provided 100% protection against lethal challenge when administered as a live vaccine. Real-time PCR analysis showed a marked increased in the levels of IL-4 and IFN-γ mRNA in immunized mice. The role that the capsule plays in SEZ pathogenicity was also explored with respect to the mechanistic design of an attenuated vaccine target. The capsule could resist complement C3 deposition on the surface of SEZ cells and aid in preventing complement-mediated opsonization and phagocytosis by cultured macrophages. These results suggest that the capsule of SEZ plays an important role in pathogenicity and may serve as a target for attenuation in vaccine development.

Attenuated Streptococcus equi ssp. zooepidemicus as a bacterial vector for expression of porcine circovirus type 2 capsid protein

Porcine circovirus type 2 (PCV2) infection and other concurrent factors is associated with post-weaning multisystemic wasting syndrome, which is becoming a major problem for the swine industry worldwide. Coinfection of Streptococcus equi ssp. zooepidemicus (SEZ) and PCV2 in swine has necessitated demand for a recombinant vaccine against these two pathogens. A recombinant SEZ-Cap strain expressing the major immunogenic capsid protein of PCV2 in place of the szp gene of acapsular SEZ C55138 ΔhasB was constructed. Fluorescence-activated cell sorting and immunofluorescence microscopy analyses indicated that the capsid protein is expressed on the surface of the recombinant strain. Experiments in mice demonstrated that strain SEZ-Cap was less virulent than the parental strain and that it induced significant anti-PCV2 antibodies when administered intraperitoneally, which is worthy of further investigation in swine.

Zebrafish (Danio rerio) as a Screen for Attenuation of Lancefield Group C Streptococci and a Model for Streptococcal Pathogenesis

Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

A novel vaccine against Streptococcus equi ssp. zooepidemicus infections: The recombinant swinepox virus expressing M-like protein

To develop a safer, more immunogenic and efficacious vaccine against Streptococcus equi ssp. zooepidemicus (SEZ) infections, the gene of M-like protein (SzP) was placed under the strong vaccinia virus promoter P28 and then inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-szp) was isolated in a non-selective medium by the co-expression of Escherichia coli LacZ gene and verified by PCR, western blotting and immunofluorescence assays. To evaluate the immunogenicity of this rSPV-szp, ICR mice were immunized with the rSPV-szp, inactivated SEZ vaccine (positive control), wild type SPV (negative control), or PBS (challenge control). All mice were intraperitoneally challenged with 5 LD 50 of homogenous ATCC 35246 strain 14 days post-vaccination. The results showed that at least 70% mice in rSPV-szp-vaccinated group were protected against homogenous ATCC 35246 challenge, the survival rate was significantly higher compared with mice in the negative control group and the challenge control group ( P < 0.001). The antibody titers of the rSPV-szp-vaccinated group were significantly higher ( P < 0.05) than the other three groups. Passive immune protection assays showed that the hyperimmune sera against M-like protein could provide mice with complete protection against challenge of ATCC 35246. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. The results suggested that the recombinant rSPV-szp provided mice with significant protection from the SEZ infections. It is a promising candidate for the vaccine development against SEZ infections.

Virulence and antigenicity of the szp-gene deleted Streptococcus equi ssp. zooepidemicus mutant in mice

Streptococcus equi ssp. zooepidemicus (SEZ) causes serious disease in pigs. M-like protein (SzP) of SEZ is a virulence factor with opsonin function. To fabricate better vaccine against SEZ, We have generated a szp-knockout strain by homologous recombination. Two fragments, upstream and downstream of the szp gene, were obtained by genome walking. The vector was then used to delete a 1090 bp segment of szp gene from a strain of SEZ (ATCC35246). The mutant strain had 1000-fold decrease in LD50 as compared with wild type, and provided 85% protection against challenge with a virulent strain when administered as a live vaccine. Semi-quantitative RT-PCR analysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. These data demonstrate that szp-knockout strain is a better candidate for vaccine development.