MA Fang , WANG Guang-yu , ZHOU Hong , MA Zhe, LlN Hui-xing, FAN Hong-jie,

1 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China

2 National Center of Meat Quality and Safety Control, Nanjing Agricultural University, Nanjing 210095, P.R.China

3 Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China

Abstract 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 eff icient 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 signif icant 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 eff icient protection against virulent SEZ. In addition, the hyperimmune sera against 102 CFU mL-1 attenuated vaccine ΔSEZ could confer signif icant protection against virulent SEZ infection in the passive immunization experiment and exhibited eff icient 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-1 via hypodermic injection. Thereafter, the mutant ΔSEZ at the dose of 102 CFU mL-1 could confer signif icant protection in mice and had less negative effects on host, which could be an effective attenuated vaccine candidate for the prevention of SEZ.

Keywords: Streptococcus equi ssp. zooepidemicus, zoonosis, attenuated vaccine, immune dose

1. lntroduction

Streptococcus equi ssp. zooepidemicus (SEZ) is a pathogen which threatens a wide range of animal species; it is responsible for upper respiratory disease, septicemia, meningitis, purulent arthritis, endocarditis, and mastitis (Holden et al. 2009; Ma et al. 2017). This bacterium is also a very serious zoonotic pathogen of humans, who will be infected through contact with infected animals and by consuming contaminated dairy products (Kuusi et al. 2006; Minces et al. 2011). In the past few decades, SEZ outbreaks have occurred in places leading to signif icant suffering and economic losses (Fulde and Valentin-Weigand 2013; Lindahl et al. 2013). Extensive and indiscriminate use of antibiotics has fasten the cycle of drug resistance and rendered most drug treatments ineffective (Walsh 2000). Several studies focused on virulence proteins of SEZ, such as antiphagocytic surface protein SzP, Fc receptors and f ibronectin-binding proteins, have been evaluated (Yi et al. 2013; Liang et al. 2018). However, it is reported that SzP contains a variable region and it is unclear what level of cross protection will be conferred by it (Walker et al. 1998). To, date, no commercial products have been progressed yet (Waller 2010). Development of a novel safe and effective vaccine would represent a major advance in overcoming SEZ.

Pathogenicity islands are groups of mobile genetic elements carrying genes for superantigen toxins and other virulence factors, which are able to facilitate the infective process of bacterial pathogens (Mir-Sanchis et al. 2012). Pathogenicity island SeseCisland_4 belongs to the restriction modif ication system, which are thought to allow bacteria to protect themselves against foreign DNA (Ma et al. 2013). Vaccines are aiming to stimulate proper immune response to clear pathogens (Moyle 2017). Attenuated vaccine is the f irst vaccine against bacterial diseases (Haesebrouck et al. 2004). Live whole microorganisms can provide protective cross-immunity against pathogens which could deliver many potential protection simulating the nature infection. It is reported that attenuated live vaccine strains could induce a strong humoral and cellular immune response, which have been shown to offer relatively good protection against infection (De la Cruz et al. 2017). Live attenuated vaccines with the capacity for mass administration may gain the greater acceptance if they can confer a solid and lasting protection against multiple strains considering that commercially vaccines are described as inducing poor and short lived immunity (Holden et al. 2014).

In swine and horse, SEZ infections lead to signif icant economic and welfare costs. In addition, SEZ may infect companion animals and even human. Gene-deletion vaccines are less likely to return to highly virulent condition (Fan et al. 2009). Thereafter, there is requirement for effective attenuated vaccines against SEZ infection. In this study, we constructed a multi-gene deletion strain to obtain an attenuated vaccine. When f ive genes were knocked out at the two f lanks of pathogenicity island SeseCisland_4, bacterial virulence decreased signif icantly. The mutant can provide signif icant protection in mice and might be a candidate for a live attenuated vaccine in future SEZ prevention efforts.

2. Materials and methods

2.1. Bacterial strains, cell and experimental animals

The wild-type SEZ strain ATCC 35246 (Sz35246) was purchased from the American Type Culture Collection (ATCC). SEZ strains CY, CT and C55138 were previously isolated from diseased animals and preserved in our laboratory (Ma et al. 2011; Wei et al. 2012). All SEZ strains were grown in Todd-Hewitt broth (THB) medium (Difco, BD, Franklin, NJ, USA) at 37°C. Bacteria were cultured to midexponential phase and washed three times in PBS and then infected animals and cells. Escherichia coli DH5a containing plasmid (p SET4s-target genes) was grown in Luria-Bertani (LB) supplemented with 50 μg mL-1spectinomycin (Sigma Aldrich, St. Louis, Mo, USA) (Ju et al. 2012). HEp-2 (ATCC?CCL-23TM) cells were purchased from ATCC and cultured in Dulbecco's modif ied Eagle's medium (DMEM; Wisent, Canada), supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. The cells were cultured in DMEM (Wisent, Canada), supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2.

Four-week-old female Institute of Cancer Research (ICR) specif ic pathogen-free mice were purchased from the Comparative Medicine Center of Yangzhou University, China. All the experiments with animals were performed in accordance to animal welfare standards and were approved by the Ethical Committee for Animal Experiments of Nanjing Agricultural University, China. The symptoms of the mice such as infected mental fatigue, loss of appetite and weight, death were documented over a period of four weeks. Animals showing signs of illnesses were humanely euthanized by cervical dislocation.

2.2. Construction of deletion mutant

The genes of pathogenicity island SeseCisland_4 were divided into three portions, RH1, RH2 and RH3, according to both locus and size (Table 1). The construction of RH1 and RH2 double genes deletion mutant strain (named as ΔSEZ) was performed as previously described (Miyoshi-Akiyama et al. 2005). Brief ly, the upstream and downstream of RH1 and RH2 were amplif ied from the chromosomal DNA of Sz35246 separately using primer combinations LA1-F/LA1-R, RA1-F/RA1-R and LA2-F/LA2-R, RA2-F/RA2-R. The upstream and downstream f lanks of RH1 and RH2 were each ligated at the Sal I and Bam HI of p SET4s thermosensitive suicide vectors to yield p SET4s-RH1 and p SET4s-RH2, respectively. The recombinant plasmid p SET4s-RH1 was electroporated into wild-type Sz35246. The plasmid pSET4s-RH1 and bacterial genome exchanged genetic fragements to obtain RH1 fragment deletion mutant ΔRH1. RH2 deletion mutant was constructed using the same process on the mutant ΔRH1 background and both RH1 and RH2 deletion mutant strain was regarded as ΔSEZ. Putative deletion mutants ΔSEZ were determined by PCR using primer combinations Check1-F/Check1-R and Check 2-F/Check2-R. All primers used in this study are listed in Table 2.

2.3. Virulence determination

The 50% lethal dose (LD50) determination was performed as previously described (Reed and Muench 1938). Sixty mice were assigned into ten groups of six each. Mice were challenged with suspension (200 μL) of either Sz35246 or ΔSEZ at dose of 5×108, 5×107, 5×106, 5×105, and 5×104colony-forming units (CFU) mL-1by intraperitoneal injection. The LD50was calculated based on the method of Bliss (Finney 1985). To compare the growth of Sz35246 and ΔSEZ, bacteria were incubated in 100 mL of THB for 12 h and the OD600was determined at each time point.

2.4. Cell adhesion assay

The cell adherence assay was performed according to a previously described method (Pracht et al. 2005). HEp-2 cells in 24-well plates were washed three times by DMEM without FBS. Semiconf luent monolayers were infected with 100 μL suspensions containing 106CFU mL-1of Sz35246 or ΔSEZ. Each 24-well plate was concentrated at 800×g for 10 min and incubated for 2 h at 37°C. Cells were then washed three times with DMEM followed by treatment with 100 μL of trypsin and 900 μL of sterile deionized water to release all bacteria. CFUs were determined by plating serial dilutions. Bacteria in DMEM without HEp-2 cells were counted as initial inoculum. The adherence rate of Sz35246 was regarded as 100%.

2.5. lmmune procedure

Sixty mice were randomly assigned into six groups of 10 each and mice were vaccinated with 200 μL of ΔSEZ at concentrations of 5×101, 5×102, 5×103and 5×104CFU mL-1via hypodermic injection, respectively. Mice immunized with 200 μL of inactive Sz35246 at a concentration of 5×107CFU mL-1in freund's adjuvant or PBS were regarded as the positive or negative control. Subsequent booster injections were given on the 14th day (Priebe et al. 2002).

Table 1 Genes in pathogenicity island SeseCisland_4

Table 2 Primers used in the study

2.6. Hyperimmune sera collection

The blood was collected at 7, 14, 21, 28 and 35 days post the initial vaccination. The collected blood was stored at room temperature for 1 h and then the coagulated blood was stored at 4°C overnight. The antisera were collected after centrifugation at 400×g for 20 min.

2.7. Active immune assay

Mice were immunized with ΔSEZ at concentrations of 5×101, 5×102, 5×103and 5×104CFU mL-1, inactive Sz35246 or PBS in 200 μL volume according to immune procedure in Section 2.5. Then mice were challenged with 200 μL of wild-type Sz35246. To determine the protection of ΔSEZ against other virulent SEZ, mice were immunized with 200 μL of ΔSEZ at concentrations of 5×102CFU mL-1or PBS and then challenged with virulent CY, CT and C55138 by intraperitoneal injection 35 days post initial immunization.

2.8. Antibody titers determination

Antibodies titers of antisera from immunized mice were detected by ELISA (Lindsay et al. 2013). The 96-well plates were coated with inactivated bacteria at the concentration of 107CFU mL-1after being sensitized by 5% (v/v) glutaraldehyde aqueous solution (Sinopharm Chemical reagent Co., Ltd., Shanghai, China) for 2 h. The coated plates were blocked with 25% bovine serum albumin (Sigma Aldrich)/PBST (PBS containing 0.05% Tween 20) at 37°C for 2 h and then washed three times with PBST. The negative control consisted of sera obtained from mice immunized with PBS; the blank control was PBST without sera. Reactions were measured at an absorbance of 450 nm (OD450) using a microtiter plate reader (Tecan Inf inite Pro, Austria). The value was calculated as: Sample OD450-Blank control group OD450)/(Negative control group OD450-Blank control group OD450). A ratio value higher than 2.0 was considered to be positive and the antibody titers were expressed as the highest dilution of antibody.

2.9. Bacterial survival in blood in vivo and in vitro

For the in vivo assay, mice were immunized with ΔSEZ at concentrations of 5×102CFU mL-1, inactive Sz35246 or PBS in 200-μL volume according to immune procedure in Section 2.5. Then mice were challenged with 200 μL of wild-type Sz35246. Bacterial burdens were enumerated in the blood at 24 and 48 h post-challenge with wild-type Sz35246. The CFUs of survival bacteria in mice immunized with PBS at 24 h post-challenge with wild-type Sz35246 were regarded as 100%.

The in vitro whole blood bactericidal assay was performed as previously described (Thammavongsa et al. 2009). Brief ly, an aliquot of 100 μL of wild-type Sz35246 at 107CFU mL-1was added to 900 μL fresh mouse blood (in 10% trisodium citrate) and 100 μL of hyperimmune sera (antibody titer was 1:12 800); samples were incubated at 37°C for 90 min with slow rotation. The positive control and negative controls consisted of inactive Sz35246 vaccine immune sera (1:800) and sera obtained from antibody-negative mice, respectively. Bacterial dilutions were plated on THB for enumeration of surviving CFUs. The CFUs of survival bacteria in negative control group were regarded as 100%. The survival rates (%) of experiment groups and positive control groups were calculated as follows: CFU mL-1in experiment groups or positive groups/CFU mL-1in negative control groups×100.

2.10. Proliferative capacity of vaccine strain

To evaluate the proliferation of vaccine strain ΔSEZ in vivo, mice were immunized with ΔSEZ at concentration of 5×102CFU mL-1in 200-μL volume according to immune procedure in Section 2.5. Blood was collected at 15, 20 and 35 days after the initial vaccination and then blood was serially diluted and plated on THB agar plates.

2.11. Passive immune protection assay

Passive immune protection assays were performed as previously described (Cao et al. 2007). Brief ly, 30 mice were randomly assigned to three groups of 10 each. In experiment groups mice were passively immunized by intravenous injection with 200 μL of hyperimmune sera against mutant strain ΔSEZ at antibody titer of 1:12 800 or hyperimmune sera against inactive Sz35246 with an antibody titer of 1:800. Mice passively immunized with sera obtained from antibody-negative mice were served as the negative control. At 24 h after the vaccination, all mice were challenged with 5×107CFU mL-1of wild-type Sz35246.

2.12. Statistical analysis

All experiments were repeated at least three times. Student's t-test and Graphpad Prism 5 Software package (GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The limit of statistical signif icance was P＜0.05.

3. Results

3.1. Deletion mutant could be an attenuate vaccine candidate

To construct the attenuated virulent strain ΔSEZ, the upstream and downstream f lanking sequences of the target genes were ligated into p SET4s and electroporated into Sz35246. Recombinant plasmid and the bacterial genome exchange genetic fragments twice through intermolecular recombination (Fig. 1-A). PCR analysis of the target gene loci conf irmed the successful construction of the multi-gene deletion mutant ΔSEZ (Fig. 1-B and C).

The LD50was performed to detect the virulence of ΔSEZ and the result showed that the virulence of ΔSEZ (LD50=1.83×106CFU mL-1) was signif icantly attenuated compared with that of Sz35246 (LD50=3.50×104CFU mL-1) (Table 3). Adhesion is the f irst step for bacterial infection and there was a 70% reduction in the adherence of ΔSEZ to Hep-2 cells comparing with that of Sz35246 (Fig. 2-A).

In addition, it is noted that the growth curve of Sz35246 and ΔSEZ in THB had no difference indicating that the difference of virulence was not caused by bacterial growth (Fig. 2-B). The result showed that the mutant strain ΔSEZ could be used as the attenuated vaccine candidate.

3.2. Vaccination dose determination

The antibody titer was up to 1:12 800 when the mice vaccinated with ΔSEZ at the dose of 102CFU mL-1. However, the antibody titers were up to 1:6 400 when the immune dose of ΔSEZ was 103and 104CFU mL-1. In addition, antibody titer of hyperimmune sera from ΔSEZ vaccinated mice was higher than that from inactive Sz35246 vaccinated mice (1:800) (Fig. 3-A). The result indicated that the protective eff iciency of ΔSEZ was better than that of inactive vaccine and appreciate vaccination dose is necessary.

Fig. 1 Diagram and conf irmation of gene deletion mutant Streptococcus equi ssp. zooepidemicus (ΔSEZ). A, the plasmid p SET4starget gene is used for deleting target genes. Upstream and downstream regions of the target genes are ligated to vector pSET4s. Recombination plasmid pSET4s-target gene and the genome of Sz35246 exchange genetic fragments to achieve gene deletion mutant. B, PCR analyses of the ΔRH1. Lane 1, marker; lanes 2-4, upstream and downstream fusion segments of ΔRH1 (2 326 bp), Sz35246 (3 781 bp) and the negative control (dd H2O); lanes 5-7, target segment RH1 (748 bp) detection in ΔRH1, Sz35246 and negative control (dd H2O). C, PCR analyses of the ΔSEZ which was derived from ΔRH1 parent. Lane 1, marker; lanes 2-4, upstream and downstream fusion segments of ΔSEZ (2 088 bp), Sz35246 (4 982 bp) and negative control (dd H2O); lanes 5-7, target segment RH2 (1 052 bp) detection in ΔSEZ, Sz35246 and negative control (ddH2O).

To further detect the protective eff iciency of mutant strain ΔSEZ, mice were vaccinated with ΔSEZ at different doses and then challenged with virulent Sz35246. The protective rate of mice immunized with ΔSEZ at dose of 102CFU mL-1were 80%, but the protective rate of mice immunized with inactive Sz35246 were 60% (Fig. 3-B). In addition, the protective rate of mice immunized with ΔSEZ at dose of 101, 103and 104CFU mL-1were 60, 80 and 60% (data not shown). The results was consistent with the results of antibody titer indicating that appropriate immune dose of attenuated vaccine could confer better protective eff iciency against virulent bacterial infection. In addition, at 24 h postinfection with wild-type Sz35246, bacterial survival rate in mice with the protection of ΔSEZ at dose of 102CFU mL-1were only 15% of that in PBS immunized mice. Importantly, mice immunized with ΔSEZ at dose of 102CFU mL-1did not exhibit bacterial loads in blood at 48 h post-infection. Meanwhile, mice immunized with inactive Sz35246 exhibited a few bacterial loads in blood at 48 h post-infection, suggesting ΔSEZ at dose of 102CFU mL-1and inactive Sz35246 could effectively eliminate virulent Sz35246 in blood in vivo (Fig. 3-C). Therefore, hyperimmune sera (1:12 800) from 102CFU mL-1of ΔSEZ vaccination mice was used in the following assays.

Table 3 Calculation of 50% lethal dose (LD50) for different strains1)

Fig. 2 Adhesion ability and growth properties of gene deletion mutant Streptococcus equi ssp. zooepidemicus (ΔSEZ) strain. A, the adherence of ΔSEZ strain was signif icantly decreased up to 70% compared with that of wild-type Sz35246. The results are depicted as the mean±SEM (n=5). ＊＊, P＜0.01. B, the growth curve of Sz35246 and ΔSEZ in Todd-Hewitt broth (THB). The results are depicted as the mean±SEM (n=3).

Fig. 3 Vaccination dose determination. A, results of antibody titer of antisera from immunized mice by ELISA. The mean log10Antibody titer is plotted against days post initial vaccination. The results are depicted as the mean±SEM (n=3). B, the survival rate of mice challenged against Sz35246 post-immunization with 102 CFU mL-1 of gene deletion mutant Streptococcus equi ssp. zooepidemicus (ΔSEZ), inactive Sz35246 and PBS. C, virulent Sz35246 survival capability in mice blood with the protection of ΔSEZ at dose of 102 CFU mL-1. The results are depicted as the mean±SEM (n=3). ＊＊, P＜0.01; ＊＊＊, P＜0.001; ns, no difference between groups.

3.3. Active immunization protection against virulent SEZ

CY, CT and C55138 are virulent SEZ strains. Mice were immunized with 102CFU mL-1ΔSEZ or PBS, and then challenged with the three virulent SEZ strains, respectively. The results showed that all mice died within one week postinfection with CY, C55138 and CT. However, the survival rate was 80% (challenged with C55138) and 90% (challenged with CY or CT) when mice were immunized with 102CFU mL-1of ΔSEZ (Fig. 4). These results showed that the mutant strain ΔSEZ could confer eff icient protective capability against virulent SEZ strains and the mutant ΔSEZ has the potential to be used as an attenuated vaccine.

3.4. Protective capability of hyperimmune sera

The survival capacity of Sz35246 in mice whole blood with hyperimmune sera (1:12 800) from ΔSEZ vaccination mice was signif icantly decreased by 70%, which indicated that hyperimmune sera against ΔSEZ could induce eff icient bactericidal activity (Fig. 5-A).

Passive immunization with mouse hyperimmune sera against ΔSEZ (1:12 800) can provide mice with signif icant protection, as a survival rate of 100% was observed. Meanwhile 70% of mice immunized with hyperimmune sera against inactive Sz35246 (1:800) survived the infection; however, all mice in the negative control group died after challenge with Sz35246 (Fig. 5-B). The results indicated that antisera from ΔSEZ vaccination mice could eliminate pathogens and protect the host from being infected.

Fig. 4 Protective capability of gene deletion mutant Streptococcus equi ssp. zooepidemicus (ΔSEZ) against virulent SEZ. The survival rate of mice challenged with virulent strain CT, CY and C55138 post-immunization with ΔSEZ at the dose of 102 CFU mL-1 or PBS.

To further determine the proliferative capacity of attenuated mutant ΔSEZ in vivo, mice were immunized by ΔSEZ at concentration of 102CFU mL-1and then survival bacteria in blood were quantif ied in plates. The results showed that mice immunized with ΔSEZ did not exhibit bacterial loads in blood (data not shown). It could be that vaccine strain ΔSEZ were eliminated quickly in blood or ΔSEZ might be diff icult to break through host barrier and transfer to blood system via hypodermic injection. The data demonstrate that ΔSEZ strain is a better candidate for vaccine development.

4. Discussion

4.1. Construction of attenuated vaccine candidate ΔSEZ

SEZ is known to cause diseases in many animal species and occasionally cause serious meningitis in humans. SEZ is an important pathogen of swine streptococcosis causing infected pig death, which is not only lead to economic losses but also threaten public health (Ma et al. 2017). It shouldn't be ignored that SEZ can lead to upper respiratory disease (presenting mainly as strangles) in horse. Extrapolation based on f igures in the Equine Quarterly Disease Surveillance Reports (Ivens et al. 2011), suggests there are approximately 1 000 outbreaks of strangles in the UK alone each year (Waller 2013). Importantly, in some cases of upper respiratory disease with clinical signs of strangles, only SEZ is found instead of co-infection with Streptococcus equi subsp. equi (SEE) and SEZ is thought to be the ancestor of SEE (Holden et al. 2009; Paillot et al. 2010). Disease caused by SEZ is believed to be more serious and more likely to occur when predisposing factors are present, such as immune compromise from underlying viral infection or stress from transportation. This study was designed to evaluate protection eff icacy of multi-gene deletion mutant ΔSEZ. Five genes in pathogenicity island SeseCisland_4 were deleted. Transposase IS1167 (SeseC_02358), plasmid replication protein Rep and AAA-class ATPase domain protein (SeseC_02367), helix-turn-helix protein (SeseC_02369) and phage integrase family integrase/recombinase (SeseC_02370) are necessary for eff icient DNA transposition, which is reported to drive evolution of virulence in pathogens and drug resistance in bacterial pathogens (Rubio-Cosials et al. 2018). The functions of conserved hypothetical protein (SeseC_02359) deleted in pathogenicity island SeseCisland_4 are unknown. The results showed that these genes might make contributions to bacterial virulence, however, the mechanisms need further exploration. In this study, ΔSEZ was found to had defect in the capacity of cell adhesion and virulence, decreasing up to 70% and 52-fold, respectively. In addition, the growth curve showed that the growth of ΔSEZ was not obviously affected and the virulence decreasing was not caused by growth properties indicating ΔSEZ could be an attenuate vaccine candidate.

Fig. 5 Immunoprotection of antisera from 102 CFU mL-1 of gene deletion mutant Streptococcus equi ssp. zooepidemicus (ΔSEZ) vaccination mice. A, the survival capability of Sz35246 in mouse whole blood with hyperimmune sera from 102 CFU mL-1 of ΔSEZ vaccination mice (1:12 800) and inactive Sz35246 vaccination mice (1:800). Negative control (NC) was blood with sera from antibody-negative mice. The results are depicted as the mean±SEM (n=5). ＊＊＊, P＜0.001; ns, no difference between groups. B, survival rate of mice in the passive immune protection assay. All mice immunized with hyperimmune sera against ΔSEZ (1:12 800) or inactivated Sz35246 (1:800). NC was mice passively immunized with sera obtained from antibody-negative mice.

4.2. Optimal immunizing dose of ΔSEZ

Conventional vaccines towards to SEZ-causing disease containing inactive whole bacteria or extracts have shown little eff icacy (Moloney et al. 2013). In addition, commercially available vaccines have generated unsatisfactory results and are mostly targeted the SeM protein of SEE and the development of an effective vaccine would represent a major asset to overcome strangles (Waller and Jolley 2007; Moloney et al. 2013). Compared with the more frequently produced protein subunit vaccines, the gene deletion attenuated vaccine possesses the advantages of allowing induction cell-mediated and antibody-mediated immunity; hence, these types of vaccine are considered highly eff icient, and in certain cases, they are able to prevent infection, in addition to, disease (Frey 2007). Both safety and immunogenicity are important standards for attenuated vaccine (Huttner et al. 2015). Therefore, an appropriate dose of attenuated vaccine is crucial for evaluating the attenuated vaccine. According to the LD50of ΔSEZ, 101, 102, 103, and 104CFU mL-1were chosen to be the candidate immunizing dose. It is found that 102CFU mL-1of ΔSEZ could cause better antibody response. The antibody titer of antiserum against 102CFU mL-1of ΔSEZ was higher than the other immunizing dose including inactive SEZ. In addition, mice with protection of mutant strain ΔSEZ at dose of 102CFU mL-1could effectively eliminate virulent Sz35246 in blood. Importantly, the mutant strain ΔSEZ at dose of 102CFU mL-1could confer effective protection against virulent Sz35246 and other virulent SEZ strains, such as CT, CY and C55138. Meanwhile, no viable ΔSEZ are detected in blood via hypodermic injection, suggesting that the attenuated strain has a lower immunological risk. These results indicate that ΔSEZ has the potential to be commercial vaccine and its optimal concentration is 102CFU mL-1.

The passive immune protection assay showed that the antiserum against 102CFU mL-1of ΔSEZ could provide the mice with 100% protective eff iciency against virulent Sz35246. The antiserum from mice immunized with 102CFU mL-1of ΔSEZ could signif icantly assist host to eliminate virulent SEZ according to bacterial survival assay in blood. These results conf irmed the eff icacy of attenuated vaccine on the perspective of the protective eff iciency of antiserum.

Unlike the other gene-deleted vaccine, this attenuated vaccine ΔSEZ was obtained through multi-gene deletion within pathogenicity island SeseCisland_4. Pathogenicity islands are considered to be mobile genetic elements which are able to encode one or more virulence and/or resistance determinants (Alibayov et al. 2014). Previous work on the Staphylococcus aureus pathogenicity island (SaPI) and 89K pathogenicity island of epidemic Streptococcus suis serotype 2 demonstrated that the pathogenicity islands regulated their excision and transfer to optimize maintenance in their original host and modulated their spread to new hosts, thus spreading the virulence factors (Zhong et al. 2014). Therefore, pathogenicity island plays important roles on bacterial virulence which might be the target to develop new attenuated vaccine. Five genes of SeseCisland_4 were deleted. It is failed to knock out whole pathogenicity island SeseCisland_4 after several attempts and the products of genes within pathogenicity island SeseCisland_4 might become necessary components for bacterial survival during the long period of evolution. However, the virulence of ΔSEZ was decreased signif icantly and it has the potential to be an attenuated vaccine. These study will provide knowledge for vaccine development which will be benef it for the control of SEZ.

5. Conclusion

In this study, a candidate attenuated vaccine strain ΔSEZ was constructed by multi-gene deletion in pathogenicity island SeseCisland_4. LD50, cell adhesion capability and growth curve, were used to detect the virulence of ΔSEZ. The results showed that the virulence of ΔSEZ was decreased signif icantly, which provide ΔSEZ with the potential to be an attenuated vaccine strain. The results of active immune assay and ELISA showed that immunizing dose of ΔSEZ at 102CFU mL-1can provide mice effective protection against virulent SEZ strain. In addition, antiserum from mice immunized with ΔSEZ at 102CFU mL-1could eliminate virulent Sz35246 eff iciently in whole blood, which indicated that antisera could provide host with effective protection against virulent SEZ. Important, the results of passive immune protection assay strengthen the protective action of antiserum. Importantly, the bactericidal effect of antiserum might have potential to disease treatment. The protective eff icacy and optimal immunizing dose of ΔSEZ on swine and horse and bactericidal potential and treatment role of antiserum on animals especially companion animal need further study.

Acknowledgements

This study was supported by the National Key R&D Program of China (2017YFD0500203), the National Natural Science Foundation of China (31672574), the Special Fund for Agro-scientif ic Research in the Public Interest, China (201403054), the Primary Research & Development Plan of Jiangsu Province, China (BE2017341), the Jiangsu Agricultural Science and Technology Innovation Fund [CX (16) 1028], and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).