Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro

Accepted Manuscript Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro L.P. Li, R. Wang, W.W. Liang, T. Huang, Y. Huang, F.G. Luo, A.Y. Lei, X. Gan, M. Chen PII:

S1050-4648(15)30042-5

DOI:

10.1016/j.fsi.2015.06.014

Reference:

YFSIM 3498

To appear in:

Fish and Shellfish Immunology

Received Date: 7 April 2015 Revised Date:

5 June 2015

Accepted Date: 11 June 2015

Please cite this article as: Li LP, Wang R, Liang WW, Huang T, Huang Y, Luo FG, Lei AY, Gan X, Chen M, Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro, Fish and Shellfish Immunology (2015), doi: 10.1016/j.fsi.2015.06.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Development of live attenuated Streptococcus agalactiae vaccine for tilapia via

2

continuous passage in vitro

3

L.P. Lia#, R. Wanga#, W. W. Lianga#, T. Huanga, Y. Huangb, F.G. Luoc, A. Y. Leia, X.

5

Gana,*, M. Chena,*

RI PT

4

6

a

7

Guangxi Academy of Fishery Sciences, Nanning 530021, China

8

b

Guangxi Center for Disease Control and Prevention, Nanning 530021, China

9

c

Liuzhou's Aquaculture Technology Extending Station , Liuzhou 545006, China

11

#

L.P. Li, R. W and W. W. Liang have contributed equally to this work.

12

*Corresponding author

13

M. Chen

14

Guangxi Academy of Fishery Sciences, Nanning, Guangxi, 530005, P.R. China

15

Email: [email protected] ; Tel: 86-771-5316547; Fax: 86-771-5316547

16

X. Gan

17

Guangxi Academy of Fishery Sciences, Nanning, Guangxi, 530005, P.R. China

18

Email: [email protected]; Tel: 86-771-5317682; Fax: 86-771-5317682

M AN U

SC

Guangxi Key Laboratory for Aquatic Genetic Breeding and Healthy Aquaculture,

AC C

EP

TE D

10

ACCEPTED MANUSCRIPT Abstract

20

Fish Streptococcus agalactiae (S. agalactiae) seriously harms the world's aquaculture

21

industry and causes huge economic losses. This study aimed to develop a potential

22

live attenuated vaccine of S. agalactiae. Pre-screened vaccine candidate strain S.

23

agalactiae HN016 was used as starting material to generate an attenuated strain S.

24

agalactiae YM001 by continuous passage in vitro. The biological characteristics,

25

virulence, and stability of YM001 were detected, and the protective efficacy of

26

YM001 immunization in tilapia was also determined. Our results indicated that the

27

growth, staining, characteristics of pulsed-field gel electrophoresis (PFGE) genotype,

28

and virulence of YM001 were changed significantly as compared to the parental strain

29

HN016. High doses of YM001 by intraperitoneal (IP) injection (1.0×109 CFU/fish)

30

and oral gavage (1.0×1010 CFU/fish) respectively did not cause any mortality and

31

morbidity in tilapia. The relative percent survivals (RPSs) of fishes immunized with

32

YM001 (1.0×108 CFU/fish, one time) via injection, immersion, and oral

33

administration were 96.88, 67.22, and 71.81%, respectively, at 15 days, and 93.61,

34

60.56, and 53.16%, respectively, at 30 days. In all tests with 1-3 times of

35

immunization in tilapia, the dosages at 1×108 and 1×109 CFU/fish displayed the

36

similar best results, whereas the immunoprotection of the dosages at 1×106 and 1×107

37

CFU/fish declined significantly (P<0.01), and 1×105 CFU/fish hardly displayed any

38

protective effect. In addition, the efficacy of 2-3 times of immunization was

39

significantly higher than that of single immunization (P<0.01) while no significant

40

difference in the efficacy between twice and thrice of immunization was seen

AC C

EP

TE D

M AN U

SC

RI PT

19

ACCEPTED MANUSCRIPT (P>0.05). The level of protective antibody elicited by oral immunization was

42

significantly higher compared to that of the control group (P<0.01), and the antibody

43

reached their maximum levels 14-21 days after the immunization but decreased

44

significantly after 28 days of vaccination. YM001 bacteria were isolated from the

45

brain, liver, kidney, and spleen tissues of fish after oral immunization and the bacteria

46

existed for the longest time in the spleen (up to 15 days). Taken together, this study

47

obtained a safe, stable, and highly immunogenic attenuated S. agalactiae strain

48

YM001; oral immunization of tilapia with this strain produced a good immune

49

protection.

50

Keywords: Tilapia; Streptococcus agalactiae; Attenuated vaccine; Oral immunization

AC C

EP

TE D

M AN U

SC

RI PT

41

ACCEPTED MANUSCRIPT 51

1. Introduction Streptococcus agalactiae (S. agalactiae) is a common aquaculture pathogen that

53

can harm different fish species, such as tilapia, Barcoo grunter (Scortum barcoo),

54

golden pompano (Trachinotus blochii), ya-fish (Schizothorax potanini), giant

55

queensland grouper (Epinephelus lanceolatus), and silver pomfret (Pampus

56

argenteus). Among them, tilapia (Oreochromis niloticus) is most seriously affected by

57

S. agalactiae-induced diseases [1-5], resulting in a high mortality and huge economic

58

losses to the aquaculture industry annually [6-8]. China is the largest tilapia farming

59

country in the world, with an output accounting for more than 40% of the world total

60

production. Since 2009, large-scale streptococcal outbreaks occurred continuously

61

with high mortality (30-90%), and more than 90% of the clinical bacterial isolates

62

were S. agalactiae [9-12].

TE D

M AN U

SC

RI PT

52

Currently, the control of S. agalactiae in aquaculture relies mainly on antibiotics.

64

However, such antibiotic usage has reportedly caused adverse effects. Accumulation

65

of antibiotics in fish can be harmful to the environment, cause potential food safety

66

hazards, and exert adverse effects on public health [13-15]. With the emergence of

67

drug tolerance and resistance, it is difficult to find drugs that can control tilapia S.

68

agalactiae diseases in China in the recent 3 years. Therefore, alternative control

69

methods, such as vaccination, are urgently needed to control S. agalactiae diseases

70

[16]. Currently, more successful S. agalactiae vaccines include the vaccine composed

71

of concentrated extracellular products (ECP) and formalin-killed whole S. agalactiae

72

cells [17-19], as well as subunit vaccine made of S. agalactiae cell surface proteins

AC C

EP

63

ACCEPTED MANUSCRIPT [20-22]. Both types of vaccines have achieved high protection efficacy against S.

74

agalactiae. However, these methods are conducted through intramuscular (IM) or IP

75

injections, which have limited their application in aquaculture, for it is laborious and

76

stressful to fish as well. By contrast, oral delivery of vaccines is more effective and

77

practical and hence has been described as the most desirable method for vaccinating

78

fishes [23]. The effects of oral administration of attenuated vaccine is better than that

79

of inactivated vaccine, mainly because of the reason that oral attenuated vaccine uses

80

live bacteria as antigen, which can greatly avoid intestinal enzymatic degradation and

81

reach the organs including spleen, kidney, etc., resulting in sustained immune

82

responses [24]. In addition, studies have shown that the orally attenuated vaccine can

83

stimulate the body to produce systemic and local mucosal immune responses

84

simultaneously [25]. To date, 2 methods have been reported for preparing attenuated S.

85

agalactiae vaccine. First, Pridgeon et al. generated an attenuated S. agalactiae strain

86

by screening against sparfloxain at continuously increased concentrations; however,

87

the obtained attenuated strain retained certain virulence to tilapia, and a strong

88

virulence more easily recovered after the removal of antibiotics; moreover, injection

89

was needed for the immunization [26]. Second, Huang et al. used live attenuated

90

Salmonella typhimurium as carrier to produce attenuated vaccine expressing S.

91

agalactiae surface antigen (Sip); however, the attenuated strain produced by this

92

method displayed a poor stability and a weak immune response, lasting for only 7

93

days by oral immunization[27]. Continuous passage in vitro is a classical method to

94

attenuate bacterial virulence and can obtain attenuated strains with significantly

AC C

EP

TE D

M AN U

SC

RI PT

73

ACCEPTED MANUSCRIPT improved stability and immunogenicity, compared to attenuated strains obtained by

96

gene knockout or screening against antibiotic stress. However, continuous passage is

97

time-consuming and labor-intensive due to the often required great number of

98

passages. To date, the attenuated S. suis strain ST171 obtained with this method in

99

1978 has been still widely used in China, with good stability, safety, and

100

immunization effectiveness [28]. However, there is no report of attenuated S.

101

agalactiae strain for fish or other hosts by naturally passaging.

SC

RI PT

95

In this study, a pre-screened vaccine candidate strain S. agalactiae HN016 was

103

continuously passaged in vitro, until obtaining a strain YM001, non-pathogenic to

104

tilapia, after 840 passages. The culture, morphology, biochemistry, serotype, genotype,

105

virulence, stability, and immunogenicity of the 2 strains were further characterized

106

and compared. Moreover, by optimizing the dose and immunization program, we

107

developed YM001 into an oral attenuated vaccine for tilapia.

108

2. Materials and methods

109

2.1 Bacterial strains and fish

TE D

EP

AC C

110

M AN U

102

S. agalactiae HN016 (serotype Ia) was isolated from an outbreak epidemical

111

disease in tilapia from Hainan, China in 2010. Our preliminary studies indicated that

112

this HN016 had good immunogenicity, can cross-protect the host from a vast majority

113

of epidemic strains in China, and thus could be a good vaccine candidate [29].

114

Non-infected Nile tilapia with average weight of 30.15±2.60 g was provided by the

115

National Tilapia Seed Farm (Nanning, Guangxi, China). Prior to experiments, the

ACCEPTED MANUSCRIPT fishes were acclimated in fiber-reinforced plastic tanks (800 L each) with a stocking

117

rate of 4 g/L under 30±4 °C for 2 weeks. The experimental fishes were confirmed to

118

be negative for bacterial infection by bacteriological analysis of the brain and kidney

119

samples. Fish in each experimental group were kept in 40L- plastic tank and all the

120

tanks were equipped with separate recirculation system with external biofilters

121

(Haisheng, China). Fish were fed twice a day with a formulated diet (Tongwei Feed

122

Company, Nanning, China). All the experiments were conducted according to the

123

principles and procedures of the Laboratory Animal Management Ordinance of China.

124

2.2 The passage of strain HN016 and virulence attenuation

M AN U

SC

RI PT

116

The strain HN016 was removed from -80 ℃ refrigerator, streaked onto a 5%

126

sheep blood agar plate, and cultured at 28 ℃ for 24 h. A single colony was then

127

picked up, inoculated into 10 mL of TSB medium, and cultivated at 25 ℃ by

128

shaking. After 12 h, 1.0 mL of bacteria was inoculated into fresh 10 mL of TSB

129

medium and cultured continuously by shaking for another 12 h. Such culture cycle

130

(passage) was repeated every 12 h, and the infection experiment was carried out after

131

each 60 passages to detect the virulence of the strain to tilapia, until that IP injection

132

of 1.0×109 CFU/fish fails to cause the death in tilapia (20 tilapias were tested each

133

time).

134

2.3

135

identification

136

AC C

EP

TE D

125

The hemolytic and

morphological

characteristics

and

bacteriological

The hemolytic activity of S. agalactiae isolates was determined by streaking the

ACCEPTED MANUSCRIPT bacteria onto 5% sheep blood agar plate, which was incubated at 28 ◦C for 24-48 h.

138

The presence and absence of a clear zone around the bacteria at the streaking sites

139

were considered hemolytic and non-hemolytic, respectively. A single colony was

140

inoculated into 100 mL of TSB and cultivated at 28 ℃ by shaking for 24 h, and the

141

culture was then used to perform the Gram staining and visualization with scanning

142

electron microscopy (SEM). The biochemical and PCR identification of the

143

attenuated strain YM001 and the parental strain HN016 were carried out according to

144

our previously published methods [12].

145

2.4 The serotyping by MLST (multilocus sequence typing) and the pulsed field gel

146

electrophoresis (PFGE) genotyping

M AN U

SC

RI PT

137

The molecular serotyping and PFGE analysis were performed as described

148

previously [12]. In MLST, 500-bp fragments of 7 housekeeping genes (adhP, pheS, atr,

149

glnA, sdhA, glcK and tkt) were amplified by PCR and sequenced. The allele numbers

150

were then assigned to each sequence and used to identify sequence types (STs) of

151

each

152

http://pubmlst.org/sagalactiae/.

153

2.5 Comparison of the virulence between YM001 and HN016

EP

TE D

147

isolate

[30].

The

allele

sequences

can

be

found

at

AC C

individual

154

The comparison of the virulence to tilapia between both YM001 and HN016

155

strains was performed by two routes, IP injection and oral gavage. Eight dosages were

156

tested in IP injection (Table 1) and two doses were used in oral gavage (1.0×109 and

157

1.0×1010 CFU/fish). Briefly, the stored HN016 and YM001 were removed from

ACCEPTED MANUSCRIPT -80 °C, streaked onto 5.0% sheep blood agar plates, and incubated at 28 °C for 24 h.

159

Single colonies were then inoculated in TSB and incubated at 28 °C for 24 h under

160

low agitation. The bacterial density (CFU/mL) was determined by plating 100 µL of

161

10-fold serially-diluted culture onto sheep blood agar plates and counting of the

162

colonies.

RI PT

158

After anesthesia by immersion into a bath of 10 mg/L benzocaine (Sigma, USA),

164

a total of 40 fishes were given HN016 or YM001 at each dose by IP injection (0.1

165

mL/fish and 20 fishes/tank, with 2 replicates). The control group was injected with 0.1

166

mL of sterile TSB. Meanwhile, in oral gavage group, each fish was administered with

167

0.5 mL of bacterial culture by oral gavage, and the control group was treated with 0.5

168

mL of TSB. The infected fishes were monitored and fed twice a day for 21 days, and

169

the bacteria were re-isolated from the brain and liver tissues of all dead fishes at the

170

end of the experiment and identified. The experiment was conducted twice.

171

2.6 Backpassage safety studies with YM001

EP

TE D

M AN U

SC

163

A backpassage safety assay was performed according to the methods of Pridgeon

173

et al. [26] with modifications. Briefly, 360 fishes were used in the backpassage safety

174

studies. They were divided into control and two vaccine groups with 180 in each

175

group, which were then divided into 12 tanks with 15 fish per tank. The fishes in tank

176

No.1 received YM001 vaccine (1.0×109 CFU/200 µL/fish) via IP injection, while the

177

control group was injected with 0.2 mL of TSB. After 48 h, 5 fishes were taken from

178

the first tank and homogenized, and the homogenate (0.2 mL) was then injected into

AC C

172

ACCEPTED MANUSCRIPT the fishes in tank No. 2. Aliquots of the homogenate were also cultured on blood agar

180

plate to determine the presence or absence of S. agalactiae. This procedure was

181

repeated eleven times with the remaining fishes, and the mortality or adverse behavior

182

or signs of disease were recorded daily for 21 days post injection.

183

2.7 Immunogenicity assay

RI PT

179

A total of 400 fishes were divided into 4 groups (100/group and 50/tank, with 2

185

replicates), in which the first 3 groups were given YM001 by IP injection, immersion,

186

and oral immunization, respectively, and the 4th group served as bland control. The

187

injection group received YM001 (1.0×108 CFU/0.1 mL/fish) via IP injection; the

188

fishes in the immersion group were soaked in YM001 culture (1.0×108 CFU/mL) at

189

28 ℃ for 30 min with aeration; and the fishes in oral administration group were fed

190

with bacteria at 1×108 CFU/fish. In the oral group, certain amount of YM001 in PBS

191

was sprayed homogeneously onto the feed mixed with adhesives, dried at room

192

temperature for 15 min, and one-time administered to the fishes. For HN016

193

challenge, the 100 fishes in each group were divided into two subgroups (50/subgroup

194

and 25/tank, with two replicates). The two subgroups were given with HN016

195

(1.0×106 CFU/fish, 100 LD50) by IP injection after 15 and 30 days of YM001

196

vaccination, respectively, and the challenged fishes were monitored and fed twice a

197

day for 15 days. Relative percent survival (RPS) was calculated as follows: RPS = {1

198

− (vaccinated mortality ÷ control mortality)} × 100. The test was repeated one time.

199

2.8 Optimization of the oral immunization doses

AC C

EP

TE D

M AN U

SC

184

ACCEPTED MANUSCRIPT To determine the optimally effective dose of YM001 in oral vaccination, the

201

bacteria at 5 different doses (1.0×105, 1.0×106, 1.0×107, 1.0×108, and 1.0×109

202

CFU/fish) were mixed with the feed, respectively, and administered to Nile tilapia for

203

oral immunization. A total of 100 fishes were used in each group, and the control

204

group was treated with equal amount of PBS. At 15 and 30 days, respectively, post

205

vaccination, 50 fishes in each group (25/tank with 2 replicates) were challenged with

206

virulent parental strain HN016 through IP injection (1.0×106 CFU/fish, 100 LD50).

207

The mortalities were recorded for 15 days after the challenge, and the presence or

208

absence of S. agalactiae in the dead fishes was determined as described above. The

209

results of S. agalactiae challenge were presented as RPS and the test was repeated one

210

time.

211

2.9 Optimization of the oral immunization program

TE D

M AN U

SC

RI PT

200

In order to determine an effective oral immunization program, tilapias were

213

divided into 4 groups and each group was further divided into 3 subgroups with 50

214

fishes in each subgroup. The fishes in the first 3 groups were orally administered with

215

feed mixed with YM001 at dosages of 1.0×107, 1.0×108, and 1.0×109 CFU/fish,

216

respectively. The fish in 4th group were orally administered with feed only and served

217

as blank control. In each group, the 3 subgroups were given with the same amount of

218

antigen once (at day 7), twice (at days 1 and 7), and thrice (at days 1, 4, and 7),

219

respectively, at an interval of 1 week. At the 30th day post the last immunization, 50

220

fishes in each subgroup (25/tank with 2 replicates) were challenged with HN016 at

221

1.0×106 CFU/fish (100 LD50) as described above. The test was repeated one time.

AC C

EP

212

ACCEPTED MANUSCRIPT 222

2.10 Tracing the attenuated strain YM001 in vivo after immunization

Before vaccination, the fishes were anesthetized with MS222. They were then

224

divided into immunization and control groups with 50 in each group. The fishes in the

225

immunization group were given YM001 (1.0×109 CFU/fish) via oral gavage

226

immunization, and the fishes in control group were treated with an equal volume of

227

TSB medium. At 0, 6, and 12 h, and 1, 3, 5, 7, 9, 11, 13, and 15 days, respectively,

228

after the immunization, 3 fishes in each group were randomly removed to dissect their

229

brain, liver, spleen, and kidney tissues under sterile conditions; the tissues were then

230

homogenized and used to isolate for bacteria by streaking onto 5% sheep blood agar

231

plates, which were observed after culture at 28 ℃ for 36 h. Meanwhile, the staining

232

and microscopic observation and PCR identification of the bacteria were performed as

233

described above.

234

2.11 Determination of the antibody levels

TE D

M AN U

SC

RI PT

223

Three fish were randomly selected from each group to evaluate the

236

YM001-specific antibody responses by ELISA at each time point after the vaccination.

237

The fishes were deeply anesthetized with MS-222, and their blood samples were

238

collected from the caudal vein. The sera were collected and stored at -80 ℃. S.

239

agalactiae YM001 was diluted to the density of 1.0×108 CFU/mL in carbonate buffer

240

(pH 9.6) and used to coat the 96-well plate at 100 µL/well. The plate was then

241

centrifuged at 200× g for 5 min and incubated at 22 ℃ for 60 min. The plate was

242

washed with PBST (0.1% Tween-20 in PBS) and blocked with 1% bovine serum

AC C

EP

235

ACCEPTED MANUSCRIPT albumin (BSA) in PBS for 2 h at 22 ℃. After washing 3 times with PBST, 100-fold

244

diluted sera were added into the wells in triplicate and incubated for 2 h at 22 ℃.

245

After washing, anti-Tilapia (Oreochromis niloticus) IgM monoclonal antibody

246

(Aquatic Diagnostic Ltd.) diluted 1:1000 in PBST was added into the wells (100

247

µL/well) and incubated for 1 h at 22 ℃. The plate was washed with PBST, added

248

with 100 µL/well of peroxidase-conjugated Goat Anti-Mouse IgG (1:1000;

249

ZSGB-BIO, China), and incubated at 22 ℃ for 1 h. After washing with PBST 3

250

times, color development was performed using the TMB kit (Tiangen, China). The

251

absorbance was read at 450 nm on a microplate reader (Bio-Rad). The serum from

252

fishes at 15 days after YM001 vaccination via IP injection served as the positive

253

control, and PBS was used as negative control. Positive results were determined when

254

the absorbance was at least twice of that of the control.

255

2.12 Statistical analysis

TE D

M AN U

SC

RI PT

243

All the data were presented as mean ± S.D. from three or four replicates and

257

analyzed by one-way ANOVA with Duncan method using software SPSS Statistics

258

17.0. The significance level was defined as P<0.05.

259

3. Results

260

3.1 Induction and characteristics of attenuated strain YM001

AC C

EP

256

261

In blood agar culture at 28 ℃, the growth of the attenuated strain YM001 was

262

significantly slower than that of the parental strain HN016. The diameter of HN016

263

colonies was up to 1 mm after 24 h of culture, and the milky-white colonies were in a

ACCEPTED MANUSCRIPT regularly circular, convex shape. However, after 24 h of culture, the YM001 colony

265

was only in a needle size, with a gray-white color; after 36 h, YM001 colonies

266

reached a diameter of up to 1 mm and appeared milky-white in color with a regularly

267

circular, convex shape (Figure 1). The HN016 strain showed a strong β-hemolytic

268

activity, whereas stain YM001 had γ-hemolysis, i.e., no hemolytic activity (Figure 1).

269

Gram staining indicated that YM001 was Gram-positive and had a greater chain

270

length, rarely showing single distribution (Figure 1), while the HN016 was

271

Gram-positive too but appeared as short chains (3-5 bacteria), with a large number of

272

single bacteria. Biochemical identification indicated that both HN016 and YM001

273

were S. agalactiae, and only the differences in leucine-arylamidase and D-ribose tests

274

(supplementary Table 1) were observed. Specific PCR identification also indicated

275

that both strains were S. agalactiae (Figure 2). Serotyping results revealed that both

276

HN016 and YM001 belonged to serotype Ia (Figure 2). The results of MLST showed

277

that both HN016 and YM001 belonged to ST-7 (Figure 2). However, PFGE analysis

278

revealed that compared with its parental strain HN016, the PFGE band pattern of

279

YM001 changed significantly (Figure 2).

280

3.2 The results of virulence and backpassage safety tests

SC

M AN U

TE D

EP

AC C

281

RI PT

264

The YM001 and parental virulent strain HN016 were administered respectively

282

into tilapia via IP injection and oral gavage, respectively. The results (Table 1) showed

283

that the LD50 (lethal dose, 50%) of injected HN016 was approximately 7.9×104

284

CFU/fish, while the mortality rates in fishes received high-dose of HN016 (1.0×109

285

and 1.0×1010 CFU/fish) by oral gavage were 90% and 95% respectively; however,

ACCEPTED MANUSCRIPT fishes infected with YM001 by either IP injection or oral gavage showed no mortality

287

or signs of disease onset, so did the fishes in control group. Among all the fishes

288

exposed to the YM001 vaccine through IP injection, no mortality or signs of disease

289

or adverse behavior was observed, and no fish died in the backpassage safety studies.

290

Attenuated strain YM001 was isolated from fish exposed to YM001 by IP in the first

291

IP and the following backpassage through IP injection of homogenates.

292

3.3 The immunogenicity of YM001

SC

RI PT

286

As shown in Table 2, for tilapia immunized with YM001 by IP injection,

294

immersion, and oral immunization, the corresponding RPSs were 96.88, 67.22, and

295

71.81%, respectively, at 15 days, and 93.61, 60.56, and 53.16%, respectively, at 30

296

days. The RPSs of the injection group at 15 and 30 days were essentially close,

297

whereas in the oral vaccination group, the RPSs at 30 days were significantly declined

298

compared with those at 15 days (P<0.05); in the immersion group, the RPSs at 30

299

days were also declined slightly, but the differences were not significant (P>0.05).

300

3.4 Optimization of the immunization dose

TE D

EP

AC C

301

M AN U

293

Tilapias were immunized with YM001 at 1.0×105, 1.0×106, 1.0×107, 1.0×108,

302

and 1.0×109 CFU/fish, respectively, and the RPSs at 15 and 30 days were recorded

303

respectively (Table 3). The corresponding RPSs at 15 days were 10.15 35.48, 50.74,

304

64.52, and 67.74%, respectively, while those at 30 days were 0.85, 10.84, 32.00,

305

53.29, and 56.24%, respectively. For both doses of 1.0×108 and 1.0×109 CFU/fish, the

306

RPSs at 15 and 30 days showed no significant difference (P>0.05); compared to

ACCEPTED MANUSCRIPT 1.0×108 and 1.0×109 CFU/fish doses, the RPSs of 1.0×106 and 1.0×107 CFU/fish dose

308

groups dropped significantly (P<0.01). The RPS of 1.0×105 PFU/fish dose was

309

extremely low, and there was almost no protection at 30 days.

310

3.5 Optimization of the immunization program

RI PT

307

As shown in table 4, the RPSs of three subgroups of the 1.0×107 CFU/fish dose

312

group were 25.10, 46.77, and 49.79%, respectively; while the protection rates of the

313

three subgroups at the dose of 1.0×108 CFU/fish were 53.75, 71.77, and 68.23%,

314

respectively, those for the 1.0×109 CFU/fish dose were 57.71, 77.50 and 73.02%,

315

respectively. At all three doses, the RPSs of the latter two subgroups were

316

significantly higher than that of the former subgroup (P<0.01). However, the RPSs

317

were not significantly different between the latter two subgroups (P>0.05).

318

3.6 Tracing of YM001 after the oral administration

TE D

M AN U

SC

311

Visual observation of the colony morphology and distribution on the blood agar

320

plate indicated that the isolated bacteria were uniform and without contamination.

321

Through growth rate, colony morphology, and staining characteristics, the isolated

322

bacteria were preliminarily identified as YM001, which was further confirmed by

323

PCR identification and PFGE analysis. The results of YM001 isolation from the brain,

324

liver, kidney, and spleen tissues at different time points after the immunization were

325

shown in Table 5. A large number of YM001 were isolated from 4 organs 6 h, 12 h,

326

and 1 day after the immunization. Generally, within the first 3 days, more numbers of

327

bacterial colonies were isolated from the body. The numbers of isolates from these

AC C

EP

319

ACCEPTED MANUSCRIPT organs at 3 to 7 days were decreased relatively, and those isolated from these organs

329

after 7 days were further reduced. However, no colony was isolated from the brain,

330

kidney, and liver at 3, 5, and 11 days, respectively, the bacterial isolation from spleen

331

remained positive at 15 days.

332

3.7 Serum antibody response

RI PT

328

ELISA results revealed that fish vaccinated with YM001 via oral route had

334

consistent absorbance readings at different time points as compared to the negative

335

control, except for that at the 7th day of 1.0×107 CFU/fish dose group (Figure 3). At

336

14 and 21 days post vaccination, the vaccinated fishes produced significantly higher

337

antibody titers compared to that of the control fishes (P<0.01). The antibody level in

338

1.0×109 CFU/fish dose group peaked at 21 days, while the antibodies in 1.0×107 and

339

1.0×108 CFU/fish dose groups reached their peak levels at 14 days. At 28 days,

340

although the antibody levels in all fishes were significantly declined compared with

341

those at 14 and 21 days, their levels were still significantly higher than those in

342

control group (P<0.05). In addition, at days 7, 14, and 21, the antibody levels in

343

1.0×108 and 1.0×109 CFU/fish dose groups were significantly higher than that in

344

1.0×107 CFU/fish dose group (P<0.05), but at 28 days, the antibody levels of the 3

345

vaccinated groups did not show significant differences (P>0.05).

346

4. Discussion

AC C

EP

TE D

M AN U

SC

333

347

Attenuated vaccines have a good immunogenicity, long period of immune

348

protection, and significant advantages especially for the development of oral and

ACCEPTED MANUSCRIPT immersion vaccines [31]. Attenuated vaccines have incomparable advantages over the

350

injection vaccines for live fish in the water. To obtain safe, stable, and well

351

immunogenic attenuated bacterial or viral vaccine strains is one of the keys for the

352

successful development of effective attenuated oral vaccines. The attenuated S.

353

agalactiae and S.iniae strains obtained by antibiotic selection and genetic engineering

354

techniques have the defects in both safety and stability (26-27, 32-33）. This study

355

used the pre-screened candidate vaccine strain HN016 to generate attenuated strain

356

YM001 by continuous passage in vitro. The phenotypic characteristics of YM001,

357

such as growth in culture, hemolysis and staining displayed significant changes

358

(Fig.1). PFGE analysis found that there were significant differences in the fingerprint

359

diagram between YM001 and HN016 strains (Fig. 2-B), suggesting the changes in the

360

genomic sequence of YM001. This result was further confirmed by the subsequent

361

sequencing and comparative analysis of the genomes, which showed that compared to

362

the HN016 genome, two DNA fragments of 5,832 and 11,116 bp, respectively, were

363

lost at two different positions in the YM001 genome, and more than 40 genes had

364

deletions or point mutations (GenBank accession number of YM001: CP011326,

365

GenBank accession number of HN016: CP011325). Therefore, the genomic changes

366

in YM001 provided the lines of evidence supporting the changes in the phenotypic

367

characteristics and virulence. In spite of these changes, the biochemical indexes

368

(supplementary Table 1）, the specific PCR confirmation（Fig. 2-A）, molecular

369

serotyping identification（Fig. 2-D）and multilocus sequence typing (MLST)（Fig. 2-C）

370

indicated that YM001 was homologous with HN016 in biochemical indexes, bacterial

AC C

EP

TE D

M AN U

SC

RI PT

349

ACCEPTED MANUSCRIPT species and serotypes and sequence type，suggesting that there are no fundamental

372

changes in the antigenicity and category evolution of YM001，which have provided a

373

good foundation for the stability of YM001. Virulence test (Table 1) showed that

374

infection of tilapia via IP injection of 1.0×109 CFU/fish or oral gavage of 1.0×1010

375

CFU/fish YM001 did not induce any mortality and morbidity in fishes, indicating that

376

even much high doses of YM001 are not pathogenic to tilapia. Meanwhile,

377

backpassage safety test showed that after 11 consecutive backpassages of YM001 in

378

tilapia (Pridgeon et al. only conducted 5 backpassages), the bacteria did not induce

379

any death or clinical symptoms in the fishes, indicating that even for the susceptible

380

tilapia, YM001 did not cause any risk of virulence recovery. In addition, genomic

381

analysis confirmed that YM001 did not contain plasmids and antibiotic resistance

382

genes transmissible genetic elements. These results confirmed that YM001 was very

383

safe for tilapia and there is no potential risk of spread of antibiotic resistance gene.

384

However, whether YM001 have virulence to other fish as well and this needs to be

385

confirmed with further experiment. A good vaccine strain must have strong

386

immunogenicity, can stimulate the body to produce high level of immune-protective

387

responses, and can simultaneously cross-protect the body from as many as

388

heterologous strains. Our previous studies indicated that except for its weak protection

389

for Ib serotype, HN016 protected well the fishes from 90% of the clinical S.

390

agalactiae isolates of Ia serotype, the dominant pathogen of tilapia in China [29]. The

391

results (table 2) of immune-protection tests in this study indicated that YM001

392

retained excellent immunogenicity of its parental strain HN016. Together, all the data

AC C

EP

TE D

M AN U

SC

RI PT

371

ACCEPTED MANUSCRIPT 393

above suggest that YM001 is safe, stable, well immunogenic, and thus suitable to be

394

developed into attenuated vaccine.

The most difficult challenge to overcome in the development of oral vaccine is

396

the period of immune-protection. Because the antigens of inactivated vaccine or

397

subunit vaccine cannot or difficult reach the immune organs, such as liver, spleen,

398

kidney, etc., in the body, they can hardly stimulate the production of systemic immune

399

responses, but can only stimulate the intestinal tract to produce local immune

400

responses, with a short-lived immune-protection, often within 7 days [27]. In addition,

401

the local mucosal immune responses elicited by inactivated antigens are relatively

402

weak, with unsatisfactory immune protection rate [34]. In this study, At 15 and 30

403

days after single immunization of tilapia with 1.0×108 CFU/fish YM001, the RPSs

404

were 71.81 and 53.16% respectively, which is absolutely superior in terms of the

405

strength and duration of immune-protection as compared to the vaccines that we

406

prepared previously using sustained-release technology with sodium alginate

407

microparticles[35]. To further improve the immune-protective effect, we optimized

408

the immunization doses and programs. Our results (Table 3) showed that in both

409

1.0×108 and 1.0×109 CFU/fish dose groups, the RPSs at 15 and 30 days after

410

immunization showed no significant difference (P>0.05); compared to 1.0×108 and

411

1.0×109 CFU/fish, the RPSs of 1.0×106 and 1.0×107 CFU/fish dose groups were

412

declined significantly (P<0.01); the RPS of 1.0×105 PFU/fish dose group was

413

extremely low. Therefore, the dose of 1.0×108 CFU/fish can be the optimal dose used

414

in the future immunization. Huang et al. showed that with same numbers of

AC C

EP

TE D

M AN U

SC

RI PT

395

ACCEPTED MANUSCRIPT immunization for oral attenuated DNA-carrier vaccine, while the RPSs of 1.0×109 and

416

1.0×108 CFU/fish dose groups were close to each other, they were all higher than the

417

that of 1.0×107 CFU/fish group [27]. Pridgeon et al. observed similar phenomenon,

418

i.e., when using attenuated S. iniae vaccine to immunize tilapia by immersion, the

419

RPSs of 1.0×107 and 1.0×106 CFU/mL dose groups reached 88 and 63%, respectively,

420

but the RPS of 1.0×105 CFU/mL group was only 13% [26]. Optimization of the

421

immunization program (Table 4) indicated that for all the 3 doses (1.0×107, 1.0×108,

422

and 1.0×109 CFU/fish), the RPSs of twice and thrice of immunization were

423

significantly higher than that of immunization once (P<0.01). However, the difference

424

between the RPSs of twice and thrice of immunization was not significant (P>0.05).

425

Therefore, twice of immunization can be used in the future practice. Similar results

426

were also reported by Huang et al., that when attenuated S. agalactiae DNA-carrier

427

vaccine was used to immunize tilapia, once or twice of booster vaccination

428

significantly improved the relative survival rate [27].

EP

TE D

M AN U

SC

RI PT

415

To further elucidate the mechanisms of attenuated YM001 vaccine, we also

430

traced the infected YM001 in vivo and assessed the antibody levels in the body post

431

immunization. The results (Table 5) showed that YM001 reached the brain, liver,

432

spleen, and kidney tissues at 6 h after the oral immunization, and multiplied into a

433

large number; 3 days later, YM001 disappeared in the brain and reduced in number in

434

liver, spleen, and kidney tissues; the number of bacteria were further reduced after 7

435

days; However, bacteria were isolated from the spleen even at 15 days after the

436

immunization. These results not only supported to certain degree the observation that

AC C

429

ACCEPTED MANUSCRIPT oral immunization of YM001 provided a relatively long term of strong

438

immune-protection (30 days), but also demonstrated that YM001 was not pathogenic

439

to tilapia. The results of ELISA (Figure 3) showed that YM001 elicited the production

440

of specific antibodies in tilapia, and the antibody levels of the 2 doses (1.0×109 and

441

1.0×108 CFU/fish) at 4 time points (7, 14, 21, and 28 days) post immunization were

442

all significantly higher than those in the control group. Meanwhile, the antibody level

443

in 1.0×108 and 1.0×109 CFU/fish dose groups was significantly higher than that in

444

1.0×107 CFU/fish group, and the antibody levels of the former 2 groups were not

445

significantly different. These data provided experimental evidences to certain extent to

446

explain the difference in RPS among the 3 doses of YM001. Huang et al. also found

447

that after oral immunization with attenuated S. agalactiae DNA-carrier vaccine, the

448

peak level of serum antibodies appeared at 14-21 days, and the antibody levels were

449

correlated with the doses and the numbers of immunization [27].

450

5. Conclusion

EP

TE D

M AN U

SC

RI PT

437

In conclusion, the present study developed a safe, stable, and well immunogenic

452

attenuated S. agalactiae strain YM001, which induced good immune-protection in

453

tilapia via oral immunization. Therefore, YM001 can be used as a novel safe and

454

efficacious vaccine to protect tilapia against S. agalactiae infections.

AC C

451

ACCEPTED MANUSCRIPT 455

Acknowledgments This work was supported by National Natural Science Foundation of China

457

(31460695), Guangxi Science and Technology Research Program (14121004-2-4),

458

Guangxi "Bagui scholar" post special funds issue (BGXZ-LFY-04) and Guangxi

459

Science Foundation (2014GXNSFBA118083 ).

TE D

M AN U

All authors have no conflicts of interest.

EP

462

Conflict of interest statement

AC C

461

SC

460

RI PT

456

ACCEPTED MANUSCRIPT References

464

[1] Jafar QA, Sameer AZ, Salwa AM, Samee AA, Ahmed AM, Al-Sharifi F.

465

Molecular investigation of Streptococcus agalactiae isolates from environmental

466

samples and fish specimens during a massive fish kill in Kuwait Bay. Pak J Biol Sci

467

2008;11:2500-4.

468

[2] Amal MN, Zamri-Saad M, Iftikhar AR, Siti-Zahrah A, Aziel S, Fahmi S. An

469

outbreak of Streptococcus agalactiae infection in cage-cultured golden pompano,

470

Trachinotus blochii (Lacepede), in Malaysia. J Fish Dis 2012;35:849-52.

471

[3] Bowater RO, Forbes-Faulkner J, Anderson IG, Condon K, Robinson B, Kong F,et

472

al. Natural outbreak of Streptococcus agalactiae (GBS) infection in wild giant

473

Queensland grouper, Epinephelus lanceolatus (Bloch), and other wild fish in northern

474

Queensland, Australia. J Fish Dis 2012;35:173-86.

475

[4] Duremdez R, Al-Marzouk A, Qasem JA, Al-Harbi A, Gharabally H. Isolation of

476

Streptococcus agalactiae from cultured silver pomfret, Pampus argenteus (Euphrasen),

477

in Kuwait. J Fish Dis 2004;27:307-10.

478

[5] Wang YT, Huang HY, Tsai MA, Wang PC, Jiang BH, Chen SC. Phosphoglycerate

479

kinase enhanced immunity of the whole cell of Streptococcus agalactiae in tilapia,

480

Oreochromis niloticus. Fish Shellfish Immunol 2014 ;41(2):250-9.

481

[6] Pereraa RP, Johnsona SK, Collinsb MD, Lewisc DH. Streptococcus iniae

482

Associated with Mortality of Tilapia nilotica×T. aurea Hybrids. J Aquat Anim Health

AC C

EP

TE D

M AN U

SC

RI PT

463

ACCEPTED MANUSCRIPT 1994;6:335-40.

484

[7] Evans JJ, Klesius PH, Shoemaker CA. Efficacy of Streptococcus agalactiae (group

485

B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath immersion

486

administration. Vaccine 2004;22, 3769–73.

487

[8] Pridgeon JW, Klesius PH. Development and efficacy of a novobiocin-resistant

488

Streptococcus iniae as a novel vaccine in Nile tilapia (Oreochromis niloticus) .

489

Vaccine 2011; 29(35):5986-93.

490

[9] Ye X, Li J, Lu MX, Deng G C , Jiang X Y , Tian Y Y , Quan Y C , Jian Q.

491

Identification and molecular typing of Streptococcus agalactiae isolated from

492

pond-cultured tilapia in China. Fish Sci 2011;77, 623-32.

493

[10] Li JR, Lu HX, Zhu JL, Wang YB, Li XP. Aquatic products processing industry in

494

China: challenges and outlook. Trends Food Sci Technol 2009;20:73-7.

495

[11] Chai JQ, Ding QL, Wang ZL, Song JY. Isolation and identification

496

ofstreptococcal bacteria isolated from tilapia. Chin J Prevent Vet Med 2002;24:18-20.

497

[12] Chen M, Li LP, Wang R, Liang WW, Huang Y, Li J, Lei AY, Huang WY, Gan X.

498

PCR detection and PFGE genotype analyses of streptococcal clinical isolates from

499

tilapia in China. Vet Microbiol 2012;159: 526-30.

500

[13] DePaola A, Peeler JT, Rodrick GE. Effect of oxytetracycline-medicated feed on

501

antibiotic resistance of gram-negative bacteria in catfish ponds. Appl Environ

502

Microbiol 1995;61:2335-40.

AC C

EP

TE D

M AN U

SC

RI PT

483

ACCEPTED MANUSCRIPT [14] Sun BG, Dang W, Sun L, Hu YH. Vibrio harveyi Hsp70: Immunogenicity and

504

application in the development of an experimental vaccine against V. harveyi and

505

Streptococcus iniae. Aquaculture 2014;418–419:144-7.

506

[15] Baquero F, Martinez JL, Canton R. Antibiotics and antibiotic resistance in water

507

environments. Curr Opin Biotechnol 2008;19:260-5.

508

[16] Darwish AM. Laboratory efficacy offlorfenicol against Streptococcus iniae

509

infection in sunshine bass. J Aquat Anim Health 2007;19:1-7.

510

[17] Evans JJ, Klesius PH, Shoemaker CA. Efficacy of Streptococcus agalactiae

511

(group B) vaccine in tilapia (Oreochromis niloticus) by intraperitoneal and bath

512

immersion administration. Vaccine 2004; 22:3769-73.

513

[18] Pasnik DJ, Evans JJ, Panangala VS, Klesius PH, Shelby RA, Shoemaker CA.

514

Antigenicity of Streptococcus agalactiae extracellular products and vaccine efficacy. J

515

Fish Dis 2005;28:205-12

516

[19] Noraini O, Sabri MY, Siti-Zahrah A. Efficacy of spray administration of

517

formalin-killed Streptococcus agalactiae in hybrid Red Tilapia. J Aquat Animal Health

518

2013;25:142e8.

519

[20] Nur-Nazifah M, Sabri MY, Siti-Zahrah A. Development and efficacy of

520

feedbased recombinant vaccine encoding the cell wall surface anchor family protein

521

of Streptococcus agalactiae against streptococcosis in Oreochromis sp.Fish Shellfish

522

Immunol 2014;37:193e200.

AC C

EP

TE D

M AN U

SC

RI PT

503

ACCEPTED MANUSCRIPT [21] Mao Z, Yu L, You Z, Wei Y, Liu Y. Cloning, expression and immunogenicity

524

analysis of five outer membrane proteins of Vibrio parahaemolyticus zj2003.Fish

525

Shellfish Immunol 2007;23:567-75.

526

[22] Cheng S, Hu YH, Jiao XD, Sun L. Identification and immunoprotective analysis

527

of a Streptococcus iniae subunit vaccine candidate. Vaccine 2010;28:2636-41.

528

[23] Johnson K, Amend DF. Efficacy of Vibrio anguillarum and Yersinia ruckeri

529

bacterins applied by oral and anal intubation of salmonids. J Fish Dis 1983;6:473-6.

530

[24] Chen WH, Garza J, Choquette M, Hawkins J, Hoeper A, Bernstein DI, Cohen

531

MB. Safety and immunogenicity of escalating dosages of a single oral administration

532

of peru-15 pCTB, a candidate live, attenuated vaccine against enterotoxigenic

533

Escherichia coli and Vibrio cholerae. Clin Vaccine Immunol 2015;22:129-35.

534

[25] Makesh M, Sudheesh PS, Cain KD. Systemic and mucosal immune response of

535

rainbow trout to immunization with an attenuated Flavobacterium psychrophilum

536

vaccine strain by different routes. Fish Shellfish Immunol 2015; 44:156-63.

537

[26] Pridgeon JW, Klesius PH. Development of live attenuated Streptococcus

538

agalactiae as potential vaccines by selecting for resistance to sparfloxacin. Vaccine

539

2013 ;31:2705-12.

540

[27] Huang LY, Wang KY, Xiao D, Chen DF, Geng Y, Wang J, et al. Safety and

541

immunogenicity of an oral DNA vaccine enco ding Sip of Streptococcus agalactiae

542

from Nile tilapia Oreochromis niloticus delivered by live attenuated Salmonella

AC C

EP

TE D

M AN U

SC

RI PT

523

ACCEPTED MANUSCRIPT typhimurium. Fish & Shellfish Immunology 2014; 38:34-41.

544

[28] Liao JR, Zhang JL, Zhen JM. Study of freeze-dried attenuated Streptococcus suis

545

ST171 vaccine. Chinese Journal of Animal and Veterinary Sciences 1983; 14:63-71

546

(in Chinese).

547

[29] Chen M, Wang R, Li LP, Liang WW, Li J, Huang Y, Lei AY, Huang WY, Gan X.

548

Screening vaccine candidate strains against Streptococcus agalactiae of tilapia based

549

on PFGE genotype. Vaccine 2012;30:6088-92.

550

[30] Evans J J, Bohnsack J F, Klesius PH, Whiting AA, Garcia JC, Shoemaker C A.

551

Phylogenetic relationships among Streptococcus agalactiae isolated from piscine,

552

dolphin, bovine and human sources: a dolphin and piscine lineage associated with a

553

fish epidemic in Kuwait is also associated with human neonatal infections in Japan.

554

Journal of Medical Microbiology 2008; 57:1369–76.

555

[31] Frey J. Biological safety concepts of genetically modified live bacterial vaccines.

556

Vaccine 2007;25:5598-605.

557

[32] Wang J, Zou LL, Li AX. Construction of a Streptococcus iniae sortase A mutant

558

and evaluation of its potential as an attenuated modified live vaccine in Nile

559

tilapia(Oreochromis niloticus). Fish & Shellfish Immunology 2014;40:392-8.

560

[33] Locke JB, Aziz RK, Vicknair MR, Nizet V, Buchanan JT. Streptococcus iniae

561

M-like protein contributes to virulence in fish and is a target for live attenuated

562

vaccine development. PloS One 2008;3:2824-9.

AC C

EP

TE D

M AN U

SC

RI PT

543

ACCEPTED MANUSCRIPT [34] Vandenberg GW. Oral vaccines for finfish: academic theory or commercial

564

reality? Anim Health Res Rev 2004; 5: 301–4.

565

[35] Chen M, Wang R, Li LL, Liang WW, Wang QH, Huang T, Li C, Li J, Gan X, Lei

566

AY, Huang WY, Luo HL.Immunological enhancement action of endotoxin-free tilapia

567

heat shock protein 70 against Streptococcus iniae. Cell Immunol 2014;290:1-9.

AC C

EP

TE D

M AN U

SC

RI PT

563

ACCEPTED MANUSCRIPT Figure legends

569

Figure 1 The growth features, hemolytic ability, Gram staining, and SEM observation

570

of S. agalactiae YM001 and HN016. A and B, the growth of HN016 and YM001 on

571

blood agar plates incubated at 28 ℃ for 36 h. The hemolysis of HN016 was evident

572

(β-hymolysis), where YM001 lost the hemolytic activity (γ-hemolysis); C,

573

microscopic observation of the Gram staining of HN016 and YM001 showed that

574

HN016 had a short chain, while the chain length of YM001 was greater; and D, The

575

morphology of HN016 and YM001 under SEM (×50,000).

576

Figure 2 The species-specific PCR, molecular serotyping, MLST, and PFGE analysis

577

of YM001 and HN016. A, in species-specific PCR identification, both HN016 and

578

YM001 showed S. agalactiae-specific 474-bp DNA bands; B, PFGE showed the

579

genome DNAs of both strains digested by SmaI restriction enzyme; C, MLST

580

analysis indicated that both HN016 and YM001 were ST-7; and D, Molecular

581

serotyping showed that both HN016 and YM001 belonged to serotype Ia. The marker

582

(M) sizes were 1000, 700, 500, 400, 300, 200, and 100 bp respectively. Lane 1 and 2

583

were HN016 and YM001 respectively.

584

Figure 3 Antibody titers in tilapia orally vaccinated with 3 different doses of YM001.

585

Data are presented as mean ± S.D. from three replicates.

AC C

EP

TE D

M AN U

SC

RI PT

568

ACCEPTED MANUSCRIPT Table 1 The virulence of YM001 and HN016 to tilapia by IP injection and oral gavage. Mortality rate (No.D/No.Tb)

Infection dose Infection route

Oral gavage Oral gavage intraperitoneal injection.

b

Nunber dead/ nunber total.

AC C

EP

TE D

a

100% (40/40) 100% (40/40) 95.00% (38/40) 85.00% (34/40) 75.00% (30/40) 55.00% (22/40) 40.00% (16/40) 10.00% (4/40) 95% (38/40) 90.00% (36/40)

0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40) 0.00% (0/40)

SC

1.0 × 109 0.5 × 109 1.0 × 108 1.0 × 107 1.0 × 106 1.0 × 105 1.0 × 104 1.0 × 103 1.0 × 1010 1.0 × 109

YM001

M AN U

IP a IP IP IP IP IP IP IP

HN016

RI PT

(CFU/fish)

ACCEPTED MANUSCRIPT

Treatment

Vaccination dose1

oral and immersion routes in tilapia.

Challenge

No. dead/

Mean mortality

Relative percent

Time

No. total2

±S.D.3

survival (RPS) ±S.D. 3

2.00±4.0a

96.88±6.25d

4.00±5.66a

93.61±9.44d

18.00±2.31b

71.81±4.01c

30/100

30.00±5.16d

53.16±6.86a

21/100

21.00±2.00bc

67.22±1.94bc

25/100

25.00±5.03cd

60.56±9.10ab

64/100

64.00±3.27e

-

64/100

64.00±5.66e

-

1.0×108

15d

2/100

Injection

1.0×108

30d

31/100

OralB

1.0×108

15d

18/100

Oral

1.0×108

30d

ImmersionC

1.0×108

15d

Immersion

1.0×108

30d

Blank control

-

15d

Blank control

-

30d

TE D

M AN U

SC

InjectionA

RI PT

Table 2 The immune-protection of YM001 administered through injection

Intraperitoneal injection.

B

Fish were fed to satiation with feeds contained vaccine YM001 for one time, and the average oral vaccination dose was 1.0×108 CFU/fish.

C

Bath immersion at 28 for 30 min. Vaccination dose for intraperitoneal injection and oral was in the unit of CFU/fish, and for bath immersion was in the unit of CFU/ml. The vaccination dose of

AC C

1

EP

A

oral treatment was average value for one fish. Total is represented by four replicate tanks of 25 fish each. Fish were challenged 15 and 30 days post-immunization by i.p. injection with 1×106 CFU/fish of S.agalactiae HN016 strain and monitored for 15 days post-challenge. 3 Means analyzed by one-way analysis of variance using the GLM procedure and Duncan's multiple range test to determine significance at P<0.05 (SPSS 17.0). Significant difference is indicated by different superscript letters. 2

ACCEPTED MANUSCRIPT Table 3 The immune-protection of different vaccination dose administered to tilapia following oral delivery. Challenge

No. dead/

Mean mortality

Relative percent

dose(CFU/fish)1

Time

No. total2

±S.D.3

survival (RPS) ±S.D.3

1.0×105

15d

58/100

58.00±2.31e

10.15±3.02a

1.0×105

30d

67/100

67.00±8.87f

0.85±15.83a

1.0×106

15d

42/100

42.00±5.16d

35.48±6.51b

1.0×106

30d

60/100

60.00±6.53ef

10.84±15.75a

1.0×107

15d

32/100

32.00±5.66c

50.74±8.96c

1.0×107

30d

46/100

46.00±4.00d

32.00±8.28b

1.0×108

15d

23/100

23.00±0.20a

64.52±4.04de

1.0×108

30d

32/100

32.00±7.30c

53.29±7.47cd

1.0×109

15d

21/100

21.00±3.83a

67.74±5.49e

1.0×109

30d

30/100

30.00±8.33bc

56.24±9.67cde

PBS control

15d

65/100

65.00±2.00ef

-

PBS control

30d

68/100

68.00±5.66f

-

SC

M AN U

TE D

1

RI PT

Vaccination

AC C

EP

Fish were fed to satiation with feeds contained different dose vaccine YM001 for one time, and the vaccination dose was average value for one fish. 2 Total is represented by four replicate tanks of 25 fish each. Fish were challenged 15 and 30 days post-immunization by i.p. injection with 1×106 CFU/fish of S.agalactiae HN016 strain and monitored for 15 days post-challenge. 3 Means analyzed by one-way analysis of variance using the GLM procedure and Duncan's multiple range test to determine significance at P<0.05 (SPSS 17.0). Significant difference is indicated by different superscript letters.

ACCEPTED MANUSCRIPT Table 4 The immune-protection of different vaccination procedure administered to tilapia following oral delivery. Vaccination

No. dead/

Mean mortality

Relative percent

Procedure1

Dose(CFU/fish)2

No. total3

±S.D. 4

survival (RPS) ±S.D.4

Once

1.0×107

50/100

50.00±5.16d

25.10±5.16 a

Once

1.0×108

31/100

31.00±5.03c

53.75±4.78 bc

Once

1.0×109

28/100

28.00±3.27 bc

57.71±7.18 c

Twice

1.0×107

36/100

36.00±8.64c

46.77±6.01b

Twice

1.0×108

19/100

19.00±3.83a

71.77±2.89d

Twice

1.0×109

15/100

Thrice

1.0×107

33/100

Thrice

1.0×108

Thrice

1.0×109

Blank control

-

SC 15.00±3.83a

77.50±5.86d

33.00±7.57c

49.79±14.45bc

M AN U

1

RI PT

Immunization

21/100

21.00±2.00ab

68.23±5.21d

18/100

18.00±5.16a

73.02±7.80d

67/100

67.00±8.87 e

-

Fish were fed with the same dose of vaccine once (at day 7), twice (at day 1 and 7),

AC C

EP

TE D

and thrice (at day 1, 4, and 7), respectively, at an interval of one week. For every vaccination, fish were fed to satiation with feeds contained vaccine YM001. 2 The vaccination dose was average value for one fish. 3 Total is represented by four replicate tanks of 25 fish each. Fish were challenged 15 and 30 days post the last immunization by i.p. injection with 1×106 CFU/fish of S.agalactiae HN016 strain and monitored for 15 days post-challenge. 4 Means analyzed by one-way analysis of variance using the GLM procedure and Duncan's multiple range test to determine significance at P<0.05 (SPSS 17.0). Significant difference is indicated by different superscript letters.

ACCEPTED MANUSCRIPT Table 5 Bacteria isolated from brain, liver, spleen, and kidney tissues after oral gavage with attenuated vaccine YM001. 0h

6h

12 h

1d

3d

5d

7d

9d

11 d

13 d

15 d

Brain

－

++++

++++

++++

－

－

－

－

－

－

－

Liver

－

++++

++++

++++

+++

++

++

+

－

－

－

Spleen

－

++++

++++

++++

+++

++

++

+

+

+

+

Kidney

－

++++

++++

++++

+++

－

－

－

－

－

－

RI PT

Tissue

AC C

EP

TE D

M AN U

SC

++++, dense growth, difficult to differentiate single colony; +++, relatively dense growth, easy to differentiate single colony; ++, less than 50 colonies; +, less than 10 colonies; and −, no colonies.

ACCEPTED MANUSCRIPT HN016

HN016

HN016

YM001

YM001

YM001

A

YM001

B

C

D

AC C

EP

TE D

M AN U

SC

Fig.1

RI PT

HN016

ACCEPTED MANUSCRIPT

A

2

1

B

C

M 1 2

CD

AC C

EP

TE D

M AN U

Fig.2

2

RI PT

M 1

SC

2 1

AC C

EP

TE D

M AN U

Fig.3

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights 1、Streptococcus agalactiae seriously harms the world's aquaculture industry. 2、Obtained a safe, stable, and highly immunogenic attenuated S. agalactiae strain

RI PT

YM001

AC C

EP

TE D

M AN U

SC

3、Oral immunization of tilapia with YM001 produced a good immune protection.

ACCEPTED MANUSCRIPT Supplementary Table 1 Biochemical characterization of YM001 and parental virulent strain HN016. D-Amygdalin Phosphatidylinositol Phospholipase C D-Xylose Arginine Dihydrolase1 Beta-Galactosidase Alpha-Glucosidase Ala-Phe-Pro Arylamidase Cyclodextrin L-Aspartate Arylamidase Beta Galactopyranosidase Alpha-Mannosidase Phosphatase

-

-

-

-

-

-

+

+

-

YM001

+

+

D-Galactose

-

-

+

-

-

D-Ribose L-Lactate Alkalinization Lactose N-Acetyl-D-Glucosamine

+

+

D-Maltose

-

-

-

-

+

Leucine Arylamidase

+

AC C

Polymixin

B Resistance

-

-

+

+

+

+

Bacitracin Resistance

+

+

Novobiocin Resistance

+

+

-

Growth In 6.5% Nacl

-

-

+

D-Mannitol D-Mannose Methyl-B-DGlucopyranoside

+

+

-

-

Pullulan

-

-

-

-

TE D

-

-

D-Raffinose O/129 Resistance

-

-

-

-

Salicin

+

+

+ + -

+ + -

Saccharose/Sucrose D-Trehalose Arginine Dihydrolase 2 Optochin Resistance

+ + + +

+ + + +

EP

L-Proline Arylamidase Beta-Glucuronidase Alpha-Galactosidase L-PyrrolidonylArylamidase Beta-Glucuronidase Alanine Arylamidase Tyrosine Arylamidase D-Sorbitol Urease

HN016

-

Characteristics

RI PT

YM001

SC

HN016

M AN U

Characteristics