The effect of intermittent hypoxia under different temperature on the immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia (Oreochromis niloticus)

Accepted Manuscript The effect of intermittent hypoxia under different temperature on the immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia (Oreochromis niloticus) Jing Wang, Dan-Qi Lu, Biao Jiang, Heng-Li Luo, Ge-Ling Lu, An-Xing Li PII:

S1050-4648(18)30225-0

DOI:

10.1016/j.fsi.2018.04.040

Reference:

YFSIM 5256

To appear in:

Fish and Shellfish Immunology

Received Date: 22 November 2017 Revised Date:

16 March 2018

Accepted Date: 19 April 2018

Please cite this article as: Wang J, Lu D-Q, Jiang B, Luo H-L, Lu G-L, Li A-X, The effect of intermittent hypoxia under different temperature on the immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia (Oreochromis niloticus), Fish and Shellfish Immunology (2018), doi: 10.1016/ j.fsi.2018.04.040. 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

The effect of intermittent hypoxia under different temperature on the

2

immunomodulation in Streptococcus agalactiae vaccinated Nile tilapia

3

(Oreochromis niloticus)

4

Jing Wang a, Dan-Qi Lu a, Biao Jiang a, Heng-Li Luo a, Ge-Ling Lu a, An-Xing Li a, b,

5

*

RI PT

1

6

a

7

Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University,

8

Guangzhou 510275, Guangdong Province, PR China;

9

b

M AN U

SC

State Key Laboratory of Biocontrol/Guangdong Provincial Key Laboratory for

Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao

National Laboratory for Marine Science and Technology, Qingdao 266235, Shandong

11

Province, PR China.

12

*Corresponding author: An-Xing Li, State Key Laboratory of Biocontrol/Guangdong

13

Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences,

14

Sun Yat-Sen University, 135 Xingang West Street, Haizhu District, Guangzhou

15

510275, Guangdong Province, PR China.

16

E–mail address: [email protected] (A.-X. Li)

AC C

EP

TE D

10

ACCEPTED MANUSCRIPT 17

ABSTRACT Dissolved oxygen (DO) and temperature are the potential immunomodulators in

19

fish and play the important roles in regulating immunity. We studied the effect of

20

intermittent hypoxia under different temperature on the immunomodulation in

21

vaccinated Nile tilapia (Oreochromis niloticus). The expression of immune-related

22

genes, enzymatic activities, histology, cumulative mortality, and S. agalactiae

23

clearance were assessed. Study conditions were intermittently hypoxic (4.0 ± 1.0

24

mg/L DO) at 30 ± 0.5°C or 35 ± 0.5°C. Interleukin-1beta (IL-1β), tumor necrosis

25

factor alpha (TNF-α) and gamma interferon (IFN-γ) mRNA expression in spleen and

26

head kidney were significantly lower in vaccinated hypoxic fish compared to the

27

vaccinated normoxic fish. Levels of heat shock protein 70 (HSP70) in tissues showed

28

an opposite tendency. Superoxide dismutase (SOD), catalase (CAT) and glutathione

29

peroxidase (GSH-Px) activities were significantly lower in vaccinated hypoxic fish.

30

Malondialdehyde levels were significantly greater under hypoxic conditions. In vitro

31

studies evaluated the effects of intermittent hypoxia at different temperatures on cells

32

of vaccinated O. niloticus. Phagocytic activity of peripheral blood leucocytes (PBLs)

33

and intracellular reactive oxygen species (ROS) production in head kidney cells were

34

significantly decreased by intermittent hypoxia at either 30°C or 35°C, while nitric

35

oxide levels in tissues cells increased significantly under hypoxic conditions. These

36

changes were well reflected by the further suppression modulation on S. agalactiae

37

clearance in vaccinated O. niloticus and higher cumulative mortality by intermittent

38

hypoxia. Taken together, intermittent hypoxia at either 30°C or 35°C could suppress

39

immunomodulation in vaccinated Nile tilapia.

SC

M AN U

TE D

EP

AC C

40

RI PT

18

41

Keywords: Streptococcus; Intermittent hypoxia; Temperature; Vaccination; Challenge;

42

Nile tilapia

ACCEPTED MANUSCRIPT 43

1. Introduction Streptococcus agalactiae is a common pathogen that causes septicemia,

45

meningoencephalitis, exophthalmia, anorexia and ascites in many fish species [1, 2].

46

High mortality rates caused by S. agalactiae have caused significant economic losses,

47

and it has become a major problem in the aquaculture industry, especially in Nile

48

tilapia (Oreochromis niloticus) [3–5]. Though S. agalactiae strains isolated from fish

49

in China have been found that are resistant to penicillin, ceftriaxone and clindamycin,

50

the misuse of antibiotics could provoke the selection of antibiotic resistant bacteria,

51

and increased the risk to the environment and human health [6, 7]. Therefore,

52

vaccines are considered a promising approach to prevent bacterial diseases, including

53

streptococcosis, in fish [8, 9]. Over these years, attempts were made to control

54

diseases by immunization, and it has been demonstrated that vaccination can

55

effectively prevent S. agalactiae infection [10–12].

M AN U

SC

RI PT

44

The immune response in fish is regulated by several factors, including

57

administration route, endogenous factors and exogenous factors, such as stocking

58

densities, pH, salinities, temperature, dissolved oxygen and water quality [13–16].

59

Among these factors, dissolved oxygen (DO) level and temperature are especially

60

important because they are closely related to disease outbreaks [17–21]. Previous

61

studies have found that the innate immunity and specific antibody titer decreased as

62

DO level decreased when fish exposed to pathogenic Edwardsiella ictaluri,

63

Aeromonas hydrophila or S. agalactiae [22–24]. In addition, the immune response

64

and tolerance to various pathogens can also be lowered by temperatures [25]. Most

65

outbreaks of streptococcosis in tilapia farms occur during warm months when water

66

temperatures were above 26°C [26]. Though S. Gallage et al. (2017) [24] had reported

67

that cumulative mortality of vaccinated fish under moderate hypoxia was significantly

68

higher than vaccinated fish under normoxic conditions, the water temperature was

69

designed at 25 ± 0.5 °C during the study, which is lower than the sensitive

70

temperature of S. agalactiae. In fact, high water temperature and/or intermittent

71

hypoxia usually occur during the hot months in tilapia farms, and these conditions

AC C

EP

TE D

56

ACCEPTED MANUSCRIPT have some effect on growth performance and innate immunity in fish [20,23].

73

However, recent studies were mainly focused on investigating the impact of high

74

temperature stress on Nile tilapia infected by S. agalactiae [18, 27] or the impact of

75

moderate hypoxia on immunomodulation in vaccinated tilapia at normal temperature

76

(~25 °C) [24]. There is little information about the influence of temperature and DO

77

on the immunomodulation in S. agalactiae vaccinated Nile tilapia, including high

78

temperature with normal oxygen, high temperature with intermittent hypoxia, or

79

intermittent hypoxia under different temperature conditions.

RI PT

72

This study is focused on assessing the immunomodulation of S. agalactiae

81

vaccinated Nile tilapia under intermittent hypoxia at different temperature conditions

82

by testing the expression profile of immune-related genes, enzymatic activities, cell

83

abilities, histology, cumulative mortality and S. agalactiae clearance. By discussing

84

the possible impact of intermittent hypoxia under different temperature on vaccinated

85

tilapia, reminding people the water environment should be attention in vaccinated fish

86

and making the vaccine efficacy optimize against S. agalactiae.

87

2. Materials and methods

88

2.1. The fish and bacterial strain

TE D

M AN U

SC

80

Tilapia (Oreochromis niloticus) juveniles were obtained from the Panyu Tilapia

90

Breeding Farm of Guangdong Province (China) and transported to the laboratory,

91

where they were reared in 1,000 L circulating tanks with constant aeration and a 29 ±

92

1°C water temperature. They were acclimatized for 30 d and fed twice daily with

93

commercial feed (Guangdong Evergreen Feed Industry Co., Ltd, China). Before

94

experiment initiation, all tested fish were confirmed to be S. agalactiae-free following

95

bacteriological examination. Approval was obtained from the Animal Ethics

96

Committee of the Life Science Institute prior to using the animals for research.

AC C

EP

89

97

S. agalactiae strain THN0901 (serotype Ia) was preserved in our lab. THN0901

98

was isolated from an intensive tilapia farm with a typical streptococcosis outbreak in

99

the Hainan province of China in 2009. Strain THN0901 has been demonstrated to be a

ACCEPTED MANUSCRIPT 100

fatal pathogen of tilapia [28].

101

2.2. Vaccine preparation This work was performed by Yongshun Biological Pharmaceutical Co., Ltd

103

(Guangzhou China). Briefly, S. agalactiae (THN0901) strains were cultured on

104

brain-heart infusion (BHI) agar culture-medium and incubated at 37 °C for 24 h.

105

Propagation of bacteria was then done by inoculating into brain-heart infusion broth

106

(BHIB) and incubated in shaker bath at 180 rpm at 28 °C for 12 h, and then overnight

107

cultured cell were diluted into 1:100 in BHIB medium. The cultured cells were grown

108

until the early stationary phase (10h) and harvested centrifugation at 10,000×g for 10

109

min at 4 °C. The cell pellet was washed repeatedly with phosphate buffered saline

110

(PBS) and then re-suspended with 0.4% buffered formalin overnight at 4°C for

111

inactivation. The formalin-killed bacteria were washed and then re-suspended in

112

sterile PBS. The suspension was streaked on BHI agar and incubated for 24 h at 30°C

113

to ensure that all S. agalactiae cells were killed and there was no contamination.

114

Finally, 70 % white oil adjuvant (Yongshun, Guangdong, China) was added to

115

improve the immune response and the final inactivated bacterial concentration was

116

3×109 CFU/ml. The prepared inactivation vaccine was stored at -4°C until use.

117

2.3. Experimental design and sample collection

SC

M AN U

TE D

EP

The experiment was conducted during August to September in 2017. Intermittent

AC C

118

RI PT

102

119

hypoxia with flow-through fresh water was delivered at either 30 ± 0.5°C or 35 ±

120

0.5°C. A total of 720 Nile tilapia juveniles (mean weight = 20.0 ± 3.0 g) were selected

121

for this study. They were acclimatized 14 d to intermittent hypoxic (4.0 ± 1.0 mg/L

122

DO) or normoxic (8.0 ± 0.5 mg/L DO) conditions. The rearing water temperature was

123

adjusted concurrently. The intermittent hypoxic groups were treated from 7:00-11:00

124

am and 18:00-22:00 pm daily. During the other times of the day, oxygen was

125

administrated like the normoxic groups. Dissolved oxygen in tanks was adjusted by

126

manipulating aeration and injecting N2 into the tanks through aerators connected with

ACCEPTED MANUSCRIPT a flow meter. Water temperature was adjusted using heating rods. Dissolved oxygen

128

and temperature in each tank were measured three times per day using an oxygen

129

meter (JPB-70A, China). The pH was 6.8 ± 0.3 and nitrite was less than 0.5 mg/L.

130

The selected fish were divided evenly into twenty-four floating glass tanks (30

131

fish/tank), and there were eight treatments in triplicate. The four control groups were

132

administrated intraperitoneally with 100 µl PBS, and the other four treatment groups

133

were administrated intraperitoneally with 100 µl of prepared vaccine at 0 d (the day

134

when we immunized the fish was defined as day 0). The eight groups were

135

administrated as follows, 30°C + normoxic (No.) + PBS, 30°C + No. + vaccination

136

(Vac.), 30°C + intermittent hypoxic (In. Hy.) + PBS, 30°C + In. Hy. + Vac., 35°C +

137

No. + PBS, 35°C + No. + Vac., 35°C + In. Hy. + PBS, 35°C + In. Hy. + Vac.. The

138

fish were fed with commercial dry feed (Evergreen, Guangdong, China) twice daily

139

and feeding was discontinued 24 h before vaccination, challenge or sampling.

M AN U

SC

RI PT

127

Fish (n = 3) were sampled randomly from each experiment group at different

141

sampling points for different purpose. After anesthetization with MS-222, spleen and

142

head kidney samples were collected at 48 h post-vaccination from each group for the

143

determination of immune-related gene expression. Organ samples were separated

144

immediately under sterile operation and stored in a Sample Protector for RNA/DNA

145

(Takara, Dalian, China) at -80°C until RNA extraction. Peripheral blood, spleens,

146

head kidneys and distal intestine were collected at 28 d post-vaccination. The serum

147

of peripheral blood without heparin was isolated by centrifugation (4000 rpm, 10 min)

148

for use in determining serum superoxide dismutase (SOD), catalase (CAT),

149

glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) levels.

150

Peripheral blood mixed with precooled heparin for use leukocytes isolation. The

151

peripheral blood leukocytes (PBLs) were isolated immediately for measuring

152

phagocytic activity. The primary spleen cells and primary head kidney cells were

153

isolated immediately from spleens and head kidneys at 28 d post-vaccination for

154

measuring respiratory burst response and nitric oxide response. Distal intestine and

155

head kidneys were obtained at 28 d post-vaccination and were fixed in 4%

156

paraformaldehyde at least 24 h for use histology observation.

AC C

EP

TE D

140

ACCEPTED MANUSCRIPT 157

2.4. Expression of immune-related genes Total RNA from collected organs was extracted using TRIzol reagent (Takara)

159

and the nucleic acid quality was measured by agarose gel electrophoresis, and the

160

concentration was determined by the absorbance at 260 nm using a Nanodrop

161

ND-2000 spectrophotometer (Quawell Technology Inc., San Jose, CA, USA). cDNA

162

was then synthesized from 1 µg total RNA using a PrimeScript RT reagent Kit with

163

gDNA Eraser (Takara). All cDNA samples were preserved at -20°C until quantitative

164

PCR was processed.

SC

RI PT

158

Real-time Quantitative PCR (RT-qPCR) for analysis of gene expression was

166

conducted in a LightCycler 480 Real Time System (Roche, Switzerland) with SYBR

167

Green Real-time PCR MasterMix (Takara). The relative expression levels of four

168

immune-related genes including interleukin-1beta (IL-1β), tumor necrosis factor alpha

169

(TNF-α), gamma interferon (IFN-γ) and heat shock protein 70 (HSP70) in spleen and

170

head kidney were examined with RT-qPCR, while β-actin, a housekeeping gene, was

171

chosen as an internal standard. All qPCR primers were designed using the software of

172

Beacon Designer 17.0 software based on the gene sequences in GenBank and are

173

listed in Table 1. The PCR cycles was conducted at 95°C for 30 s, followed by 40

174

cycles, each consisting of at 95°C for 5 s, and 60°C for 30 s. Each sample was run in

175

triplicate. Additionally, dissociation-curve analysis was performed and showed a

176

single peak in all cases. The relative expression was analyzed using the 2

177

method according to Livak and Schmittgen [29].

178

2.5. Non–specific immune parameters assay

TE D

EP

– ∆∆Ct

AC C

179

M AN U

165

SOD, CAT, GSH-Px activities and MDA content in serum were measured using

180

assay kits (Nanjing Jiancheng Ins., China) according to the manufacturer’s

181

instructions. Details of the procedures were described by the previous methods [30–

182

32].

183

2.6. Phagocytosis activity, respiratory burst and nitric oxide assay

ACCEPTED MANUSCRIPT 184

2.6.1. Isolation of PBLs, spleen and head kidney cells Peripheral blood was collected and PBLs were isolated immediately as described

186

by Ding et al. (2012) [33]. Briefly, the blood was diluted with an equal volume of

187

RPMI 1640 (GE, USA) medium. The suspension was transferred to the surface of

188

Ficoll-Paque PLUS (GE, USA) with a pipette, then isolated by centrifugation at 1600

189

rpm for 45 min at 19°C. PBLs collected at the interphase between the first and second

190

gradient interfaces. Then cells were removed and washed by resuspension five times

191

with equal volumes of RPMI 1640. The leucocytes were then resuspended with tissue

192

culture medium (TCM), which was prepared from RPMI 1640 medium supplemented

193

with 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO, USA) and 1%

194

streptomycin/penicillin (Sigma). The quality and quantity of isolated leucocytes were

195

tested using a cell counter (Cellometer AUTO 1000, USA) and then adjusted to 1 ×

196

10 6 cells/ml for using to phagocytosis activity analysis.

M AN U

SC

RI PT

185

Primary spleen cells and primary head kidney cells were obtained from tilapia as

198

previously described by Peng et al. (2016) [34]. The spleens and head kidneys of

199

individual fish were aseptically removed and immediately placed in TCM. The

200

spleens and head kidneys were then ground into pieces with 180 °C-treated frosted

201

glass slides in TCM and were filtered through a 100-µm cell strainer. The obtained

202

cells were washed with RPMI 1640 twice, and resuspended in TCM. The quality and

203

quantity of cells were tested as above for use in respiratory burst response and nitric

204

oxide response.

205

2.6.2. Phagocytosis activity

AC C

EP

TE D

197

206

S. agalactiae strain (THN0901) was killed as described in 2.2. Inactivated bacteria

207

(1 × 109 CFU/ml) were resuspended with TCM. The bacteria were incubated 1 h with

208

1 mg/ml fluorescein isothiocyanate (FITC) at 30°C. After washing five times with

209

RPMI 1640, 200 µl FITC-bacteria were added into the prepared PBLs (800 µl, 1 × 10

210

6

211

min and washed three time with RPMI 1640. The precipitate was incubated 5 min

cells/ml) and cultured 1 h at 28°C. Then they were centrifuged at 500 rpm for 10

ACCEPTED MANUSCRIPT with 1 ml 0.125% trypan blue to quench extracellular fluorescence. After washing

213

three times with PBS, the precipitate resuspended with 0.9% NaCl. The phagocytosis

214

activity of resuspended PBLs was analyzed by flow cytometry (FCM) (FC500, USA).

215

Side-scatter (SSC) and forward-scatter (FSC) parameters were used to determine cell

216

granularity and cell size, respectively.

217

2.6.3. Respiratory burst assay

RI PT

212

Intracellular superoxide production was measured using a nitro blue tetrazolium

219

assay (NBT assay) as described by Peng et al. (2016) [34]. Briefly, primary spleen

220

cells and primary head kidney cells were isolated from three fish of each group

221

respectively. A 100 µl amount of cells suspension was immediately seeded into

222

96-well transparent plates at a density of 1 × 106 cells/ml. The plate was incubated for

223

5 min and then centrifuged at 1500 rpm for 5 min at room temperature. The

224

supernatant was replaced immediately with 100 µl PBS + 100 µl NBT (2 mg/ml,

225

Sigma), 100 µl inactive S. agalactiae (1 × 109 CFU/ml) + 100 µl NBT, and 100 µl

226

phorbol ester (PMA) (100 ng/ml, Sigma) + 100 µl NBT, respectively. The production

227

in NBT caused by cells alone was used as a base line. The ROS production caused by

228

PMA was used as the positive control. At the end of the 1 h incubation at room

229

temperature, the non-reduced NBT was removed using 70% methanol. The cell button

230

was air dried and the reduced NBT was dissolved using 120 µl KOH (2 M). A total of

231

140 µl DMSO (Sigma) was added to dissolve the blue crystals that had formed in the

232

cytoplasm. Then the OD values were read at 630 nm using a microplate reader

233

(TECAN infinite M200 Pro Nanoquant, Swizerland).

234

2.6.4. Nitric oxide assay

AC C

EP

TE D

M AN U

SC

218

235

Primary spleen cells and primary head kidney cells were isolated immediately

236

from three fish of each group respectively as described in 2.6.1. Nitrite concentration

237

in tissue cells was measured as an indicator of NO production according to the Griess

238

reaction using a NO determination kit (Beyotime, Jiangsu, China) as described by Bai

ACCEPTED MANUSCRIPT et al. (2013) [35]. Briefly, 100 µl isolated supernatant were immediately seeded in

240

96-well transparent plates at a density of 1 × 106 cells/ml. Then they were mixed with

241

50 µl Griess reagent Ι and 50 µl Griess reagent ΙΙ, and then the absorbance was read at

242

550 nm using a microplate reader (TECAN, Swizerland).

243

2.7. Distal intestine and head kidney histology

RI PT

239

The sections of tissues were made according to standard histological techniques as

245

described by Su et al. (2017) [36]. Fixed samples of the distal intestine and head

246

kidney were routinely dehydrated in ethanol, equilibrated in xylene and embedded in

247

paraffin, and cut into 4 µm thick sections on a rotary microtome RM2135 (Leica,

248

Wetzlar, Germany). The sections were stained with hematoxylin and eosin (HE) for

249

histology observation. Blinded evaluation of the histological samples was performed

250

using an optical microscope DFC495.

251

2.8. Experimental challenge

M AN U

SC

244

S. agalactiae (THN0901) strain was cultured as described in 2.2. The median

253

lethal dose (LD50) in a challenge test was determined from a preliminary experiment

254

(data not shown). The final bacterial concentrations were confirmed by plating

255

ten-fold serial dilutions onto BHI agar medium.

EP

TE D

252

Fish (n=16) from each tank were infected by intraperitoneal injection with live S.

257

agalactiae (THN0901, 2×107 CFU/fish) and returned to their original treatment tanks

258

at 29 d following vaccination. 384 fish were used with 48 per group (triplicate tanks).

259

Mortality was recorded for 14 d after challenge and cumulative mortality was

260

statistically calculated. Additionally, the re-isolation of the S. agalactiae strain from

261

all dead fish was confirmed. The relative percentage survival (RPS) was calculated

262

using the following formula:

263

RPS = [1 − (%Mortality in vaccinated group/%mortality in control group)] × 100

264

2.9. Clearance of S. agalactiae

AC C

256

ACCEPTED MANUSCRIPT The ability of S. agalactiae clearance in tissues was measured as described by S.

266

Gallage et al. (2017) [24]. Briefly, fish (n=3) were randomly sampled to collect blood,

267

spleen, head kidney and brain samples from each experimental group at 1 d and 3 d

268

post-challenge. After anesthetization with MS-222, blood was collected from the

269

caudal vein using a 1 ml sterile syringe, mixed with precooled heparin to prevent

270

clotting and kept on ice. Spleen, head kidney and brain were aseptically removed and

271

immediately placed in 5 ml RPMI 1640 medium RPMI 1640 without antibiotic

272

supplement. Samples were kept on ice until use for bacteria re-isolation.

RI PT

265

A 500 µl sample of heparinized blood was added to 500 µl of distilled water to

274

disrupt the cells and release any surviving bacteria within the cells. The suspension

275

was serially diluted in PBS. A 100 µl sample of each dilution was plated on BHI and

276

incubated at 37°C for 24 h prior to CFU determination. CFU was expressed as mean

277

CFU (n=3 from each group at each sampling point) and the bacteria count was given

278

as CFU/ml of blood. Spleen, head kidneys and brains (100 mg from each) were

279

ground into pieces and serially diluted in PBS, separately. A 100 µl sample of each

280

dilution was plated on BHI and incubated at 37°C for 24 h, CFU were calculated as

281

above.

282

The CFU /ml of blood = No. of CFU counted on plate × dilution factor

283

The CFU /g of tissue = No. of CFU counted on plate × dilution factor

284

2.10.

M AN U

TE D

EP

Statistical analysis

AC C

285

SC

273

Each group in the present experiment was performed in triplicate. Data were

286

expressed as means ± SD. The significant values were calculated using ANOVA and

287

Ducan’s test by SPSS 19.0 (IBM, USA). The results were considered as significant at

288

p < 0.05.

289

3. Results

290

3.1. Expression of immune-related genes

ACCEPTED MANUSCRIPT 291

In both tissues, the expression levels of IL-1β, TNF-α, IFN-γ, and HSP70 were all

292

significantly up-regulated (p < 0.05) in vaccinated fish compared to non-vaccinated

293

fish at 48 h post-vaccination at either 30°C or 35°C. In the spleen (Fig. 1), IL-1β (Fig. 1 A), TNF-α (Fig. 1 B) and IFN-γ (Fig. 1 C)

295

mRNA levels were all significantly down-regulated (p < 0.05) in vaccinated hypoxic

296

fish compared to vaccinated normoxic fish at either 30°C or 35°C. However, HSP70

297

(Fig. 1 D) mRNA levels in vaccinated hypoxic groups were significantly up-regulated

298

(p < 0.05) compared to vaccinated normoxic fish at both temperatures. In the head

299

kidney (Fig. 2), the expression levels of IL-1β (Fig. 2 A), TNF-α (Fig. 2 B), IFN-γ

300

(Fig. 2 C) and HSP70 (Fig. 2 D) were similar to the expression levels in spleen.

301

3.2. Non–specific immune parameters assay

M AN U

SC

RI PT

294

The SOD, CAT, GSH-Px activities and MDA content in the serum of each group

303

at 28 d post-vaccination were studied. Fig. 3 shows that SOD (Fig. 3 A), CAT (Fig. 3

304

B), GSH-Px (Fig. 3 C) activities were significantly higher (p < 0.05) in the

305

vaccination groups than the non-vaccination groups. At 30°C or 35°C, SOD (Fig 3 A),

306

CAT (Fig 3 B) and GSH-Px (Fig. 3 C) activities in vaccinated fish maintained under

307

hypoxic conditions decreased significantly (p < 0.05) compared to vaccinated fish

308

under normoxic conditions. MDA (Fig. 3 D) content increased significantly (p < 0.05)

309

in hypoxic fish, and no significant differences (p < 0.05) were found between

310

vaccinated hypoxic group and non-vaccinated hypoxic group at either 30°C or 35°C.

311

3.3. Phagocytosis activity

EP

AC C

312

TE D

302

To elucidate the possible impact of intermittent hypoxia at different temperatures

313

on cells of the vaccinated fish, phagocytic activity was subsequently determined by

314

flow cytometry at 28 d post-vaccination. Fig. 4 A showed that PBLs with

315

FITC-bacteria were gated (P) on forward and side scatter (FS-SS) do plot, Q

316

demonstrated the bacteria distribution (no showed). Phagocytic percentages from

317

histograms (Fig. 4 A30 – D35) were statistically calculated and showed by bar graph

ACCEPTED MANUSCRIPT (Fig. 4 B). Fig. 4 B shows that the phagocytic percentage of PBLs towards S.

319

agalactiae increased significantly (p < 0.05) in the vaccination groups compared to

320

the PBS groups. The phagocytic capacity of PBLs decreased significantly (p < 0.05)

321

in the intermittent hypoxic and vaccinated groups compared to normoxic and

322

vaccinated groups at 30°C or 35°C.

323

3.4. Respiratory burst assay

RI PT

318

The effects of S. agalactiae on ROS production in tilapia primary spleen cells and

325

head kidney cells are shown in Fig. 5. In primary spleen cells, a significantly lower (p

326

< 0.05) ROS production was observed in vaccinated hypoxic fish compared to

327

vaccinated normoxic fish caused by S. agalactiae at 35°C (Fig. 5 B), but significant

328

differences were not found between these two groups at 30°C (Fig. 5 A). In primary

329

head kidney cells, a significant decrease (p < 0.05) was found in vaccinated hypoxic

330

fish compared to vaccinated normoxic fish caused by S. agalactiae at either 30°C (Fig.

331

5 C) or 35°C (Fig. 5 D).

332

3.5. Nitric oxide assay

TE D

M AN U

SC

324

The possibility that intermittent hypoxia under different temperature induced the

334

changes of nitric oxide levels was investigated in primary spleen cells and head

335

kidney cells. Tilapia spleen and head kidney cells were stimulated with S. agalactiae.

336

Nitric oxide levels in spleen cells and head kidney cells are shown in Fig, 6 A and Fig.

337

6 B, respectively. In both tissue cells, nitric oxide levels increased significantly (p <

338

0.05) in vaccinated hypoxic fish compared to vaccinated normoxic fish at either 30°C

339

or 35°C.

340

3.6. Distal intestine and head kidney histology

AC C

EP

333

341

The results of HE staining in distal intestine and head kidney of vaccinated fish

342

under different DO and temperature conditions at 28 d post-vaccination show in Fig.7

343

(the tissue sections of non-vaccinated fish under different conditions do not showed

ACCEPTED MANUSCRIPT here because no significant differences were found between vaccinated fish and

345

non-vaccinated fish under the same treatment). In distal intestine, villus showed some

346

degree of shedding in vaccinated hypoxic fish at 30°C (Fig. 7 In-B) and 35°C (Fig. 7

347

In-D) compared to vaccinated normoxic fish at 30°C (Fig. 7 In-A) and 35°C (Fig. 7

348

In-C). In head kidney, intercellular hyperplasia and healthy red cells decreasing were

349

found in vaccinated hypoxic fish at 30°C (Fig. 7 HK-B) and 35°C (Fig. 7 HK-D)

350

compared to vaccinated normoxic fish at 30°C (Fig. 7 HK-A) and 35°C (Fig. 7 HK-C).

351

Furthermore, substantial hemosiderin was found in vaccinated normoxic fish and

352

vaccinated hypoxic fish in head kidney at 35°C.

353

3.7. Cumulative mortality following challenge

M AN U

SC

RI PT

344

To assess the protective efficacy of vaccine in vaccinated fish under different

355

temperature and dissolved oxygen conditions, tilapias were separately immunized

356

with PBS and inactivation S. agalactiae vaccine on day 0, and then challenged with S.

357

agalactiae on day 29. Fig. 8 shows the percentage cumulative mortality of tilapia.

358

Tilapia mortalities occurred in large quantities from 1 to 7 d post-challenge, and

359

vaccinated fish had a significantly lower (p < 0.05) cumulative mortality compared to

360

non-vaccinated fish. The final mortality of each group was 54.17 ± 2.95 % (30°C

361

normoxic control), 10.42 ± 2.95 % (30°C normoxic vaccinated), 58.33 ± 5.89 %

362

(30 °C intermittent hypoxic control), 14.58 ± 2.95 % (30 °C intermittent hypoxic

363

vaccinated), 68.75 ± 5.10 % (35°C normoxic control), 18.75 ± 5.10 % (35°C

364

normoxic vaccinated), 72.92 ± 5.89 % (35°C intermittent hypoxic control) and 27.08

365

± 2.95 % (35°C intermittent hypoxic vaccinated), respectively. The RPSs of normoxic

366

vaccinated fish at 30°C, intermittent hypoxic vaccinated fish at 30°C, normoxic

367

vaccinated fish at 35°C and intermittent hypoxic vaccinated fish at 35°C compared to

368

the PBS groups were 81.02 ± 4.58%, 75 ± 4.08%, 72.32 ± 8.20% and 62.47 ± 6.05%,

369

respectively.

370

3.8. Clearance of S. agalactiae in blood, spleen, head kidney and brain

AC C

EP

TE D

354

ACCEPTED MANUSCRIPT During the challenge period, the overall bacterial burden was significantly higher

372

(p < 0.05) in the PBS groups compared to the vaccination groups (Table 2). In blood,

373

culturable S. agalactiae cells were not detected in vaccinated fish at 3 d

374

post-challenge. At 1 d post-challenge, the bacterial burdens in vaccinated fish spleen

375

and head kidney were significantly higher (p < 0.05) in hypoxic groups compared to

376

normoxic groups either at 30°C or 35°C. Furthermore, spleen in non-vaccinated fish

377

kept under 35°C and hypoxic condition contained the highest bacterial burden at 1 d

378

post-challenge.

379

4. Discussion

SC

RI PT

371

Both temperature and DO at the time of vaccination are recognized as important

381

factors in the development of protective immunity in ectothermic vertebrates [18, 22,

382

24]. To address the potential role of intermittent hypoxia at different temperatures on

383

immune adjustment in vaccinated Nile tilapia, in vivo and in vitro experiments were

384

studied. The present study showed that IL-1β, TNF-α and IFN-γ mRNA levels were

385

all strongly down-regulated in vaccinated hypoxic fish at either 30°C or 35°C. This

386

suggests that intermittent hypoxia at either 30°C or 35°C may have an inhibitory

387

effect on the expression of important pro-inflammatory genes (IL-1β, TNF-α and

388

IFN-γ). These results are also supported by recent studies, demonstrating that IL-1β

389

transcription decreases in response to acute hypoxia in Nile tilapia [37] and long-term

390

hypoxia either reduces or delays the expression of IL-1β, TNF-α and IFN-γ genes in

391

Atlantic salmon (Salmo salar L.) [38].The modulated effects by inhibitory

392

immune-related genes were further detected in primary cells from tilapia peripheral

393

blood, spleen and head kidney, including restraining phagocytosis, respiratory burst

394

activities and enhancement of nitric oxide production in vaccinated hypoxic fish at

395

both temperatures in the present study. These changes in vitro are closely related to

396

the functions of pro-inflammatory cytokines because IL-1β can increase yeast

397

phagocytosis by recruiting and proliferating head kidney leukocytes [39], TNF-α can

398

result in priming of the respiratory burst of the peritoneal exudate and head kidney

AC C

EP

TE D

M AN U

380

ACCEPTED MANUSCRIPT leukocytes [40], and stimulation of peripheral blood leukocytes with IFN-γ-related

400

protein resulted in the activation of IFN-γ receptor and marked induction of inducible

401

nitric oxide synthase gene expression [41], so these three immune-related genes could

402

lead to changes in phagocytosis, respiratory burst and nitric oxide production by

403

above ways. Additionally, HSP70, a member of the HSP protein family, has powerful

404

immune regulatory effects [42, 43]. Under stress such as hypoxia, the anti-apoptosis

405

and synergetic immunity of HSP70 can be strengthened in order to protect cells from

406

environmental stressors [44, 45], this may be the reason why HSP70 mRNA levels

407

were strongly up-regulated in vaccinated hypoxic fish at both temperatures in present

408

study.

SC

RI PT

399

In non-specific immune system, oxygen consumption is necessary to maintain the

410

NADPH oxidase level in phagocytes in order to activate biochemical reactions that

411

generate ROS [46]. SOD, CAT and GSH-Px, as important antioxidants, play vital

412

roles on transferring ROS for protecting membranes and DNA from damage [47,48].

413

However, teleost fish have a low capacity for regulation of internal levels of dissolved

414

oxygen or temperature by adjusting their physiological, biological mechanisms or

415

behavior [49]. When fish are under hypoxic conditions, the immune system may

416

experience a similar phenomenon because their functions may be affected by the level

417

of hypoxia [24]. Furthermore, substantial hemosiderosis were found in head kidney of

418

vaccinated fish at 35°C in present study. Hemosiderin often forms after hemorrhage.

419

When blood leaves a ruptured blood vessel, the red blood cell dies and the

420

hemoglobin of the cell is released into the extracellular space [50]. In this study,

421

excessive hemosiderin to accumulate may be for red blood cell destruction under

422

hypoxic and high temperature condition. This suggests that the ability of transporting

423

oxygen by red blood cells could be weakened in vaccinated fish under these

424

conditions. Therefore, the activities of SOD, CAT and GSH-Px may be weak driven

425

by lower dissolved oxygen level and/or high temperature. Meanwhile, as the

426

breakdown product of lipid peroxides, increasing MDA content due to hypoxia may

427

have strong cytotoxicity [51]. These negative effects could further suppress the

428

phagocytic activity in cells of the immune system.

AC C

EP

TE D

M AN U

409

ACCEPTED MANUSCRIPT The pathogen clearance study demonstrated that a higher blood and tissue

430

bacterial burden was present in vaccinated fish under hypoxic conditions. This may

431

relate to the lower phagocytic capacity and ineffective vaccine absorption. The villus

432

showed shedding in vaccinated hypoxic fish in the present study and this can directly

433

affect the absorption of hindgut to antigen, which had been found that the main site of

434

antigen absorption was in hindgut in the study of teleost [52]. Furthermore, a higher

435

percentage of cumulative mortality also indicates that fish were not getting the

436

expected level of protection from vaccination when vaccinated fish kept at

437

intermittent hypoxic under different temperature condition, and the suppression

438

modulation by intermittent hypoxia under different temperature may affect local as

439

well as systemic immunoreaction.

M AN U

SC

RI PT

429

In conclusion, our findings indicate that intermittent hypoxia at either 30°C or

441

35°C could suppress immune response in vaccinated Nile tilapia. The occurrence of

442

intermittent hypoxia under different temperatures helps to explain why fish are not

443

getting the expected level of protection from vaccination.

444

Acknowledgments

The Oceanic and Fishery Adminictration of Guangdong Province (2015, 2016).

EP

446

This work was funded by Special Science Projects for Fish Diseases Control from

AC C

445

TE D

440

ACCEPTED MANUSCRIPT References

448

[1] L. Liu, Y. Li, R. He, X. Xiao, X. Zhang, Y. Su, et al., Outbreak of Streptococcus agalactiae

449

infection in barcoo grunter, Scortum barcoo (McCulloch & Waite), in an intensive fish farm

450

in China, J. Fish. Dis. 37 (2014) 1067–1072.

451 452

RI PT

447

[2] J.W. Pridgeon, P.H. Klesius, Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin, Vaccine 31 (2013) 2705–2712. [3] R.T. Chideroli, N. Amoroso, R.M. Mainardi, S.A. Suphoronski, et al., Emergence of a new

454

multidrug-resistant and highly virulent serotype of Streptococcus agalactiae in fish farms

455

from Brail, Aquacultrue 479 (2017) 45–51.

SC

453

[4] R.O. Bowater, J. Forbes-Faulkner, I.G. Anderson, K. Condon, B. Robinson, F. Kong, et al.,

457

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

458

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

459

Australia, J. Fish Dis. 35 (2012) 173–186.

M AN U

456

[5] C.M. Delannoy, R.N. Zadoks, F.A. Lainson, H.W. Ferguson, M. Crumlish, J.F. Turnbull,

461

M.C. Fontaine, Draft genome sequence of a nonhemolytic fishpathogenic Streptococcus

462

agalactiae strain, J. Bacteriol. 194 (2012) 6341–6342.

TE D

460

[6] C. Chu, P.Y. Huang, H.M. Chen, Y.H. Wang, I.A. Tsai, C.C. Lu, C.C. Chen, Genetic and

464

pathogenic difference between Streptococcus agalactiae serotype Ia fish and human isolates.

465

BMC Microbiol. 16 (2016) 175.

EP

463

[7] S. Abutbul, A. Golan-Goldhirsh, O. Barazani, D. Zilberg, Use of rosmarinus officinalis as a

467

treatment against Streptococcus iniae in tilapia (Oreochromis sp.). Aquaculture 238 (2004)

468 469 470

AC C

466

97–105.

[8] B. Magnadottir, Immunological control of fish diseases, Mar. Biotechnol. 12 (2010) 361– 379.

471

[9] G.J. Liu, J.L. Zhu, K.M. Chen, T.T. Gao, H.C. Yao, Y.J. Liu, W. Zhang, C.P. Lu,

472

Development of Streptococcus agalactiae vaccines for tilapia. Dis Aquat. Organ. 170 (2016)

473

163–170.

474

[10] L.G. Pretto-Giordano, E.E. Muller, P. Klesius, V.G. da Silva, Efficacy of an experimentally

475

inactivated Streptococcus agalactiae vaccine in Nile tilapia (Oreochromis niloticus) reared in

ACCEPTED MANUSCRIPT 476

Brazil, Aquacult Res. 41 (2010) 1539–1544.

477

[11] D.J. Pasnik, J.J. Evans, P.H. Klesius, A microwave–irradiated Streptococcus agalactiae

478

vaccine provides partial protection against experimental challenge in Nile tilapia,

479

Oreochromis niloticus, World J. Vaccines 4 (2014) 184–189. [12] M. Firdaus-Nawi, S.M. Yusoff, H. Yusof, S.Z. Abdullah, M. Zamri–Saad, Efficacy of feed–

481

based adjuvant vaccinie against Streptococcus agalactiae in Oreochromis spp. in Malaysia,

482

Aquacult Res. 45 (2013) 87–96.

RI PT

480

[13] H. Van Loveren, J.G. Van Amsterdam, R.J. Vandebriel, T.G. Kimman, H.C. Rümke, P.S.

484

Steerenberg, J.G. Vos, Vaccine-induced antibody responses as parameters of the influence of

485

endogenous and environmental factors, Environ. Health Perspect. 109 (2001) 757–764.

487 488 489

[14] L.A. Levin, D.L. Breitburg, Linking coasts and seas to address ocean deoxygenation, Nat. Clim. Change 5 (2015) 401–403.

M AN U

486

SC

483

[15] M. Cannas, P. Domeniȧ, C. Lefranҁois, The effect of hypoxia on ventilation frequency in startled common sole Solea solea, J. Fish Biol. 80 (2012) 2636–2642. [16] S. Gallage, T. Katagiri, M. Endo, K. Futami, M. Endo, M. Maita, Influence of moderate

491

hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus (Nile

492

tilapia), Fish Shellfish Immunol. 51 (2016) 271–281.

TE D

490

[17] E. Soto, N. Brown, Z. O. Gardenfors, S. Yount, F. Revan, S. Francis, M.T. Kearney, A.

494

Camus, Effect of size and temperature at vaccination on immunization and protection

495

conferred by a live attenuated Francisella noatunensis immersion vaccine in red hybrid

496

tilapia, Fish Shellfish Immunol. 41 (2014) 593–599.

EP

493

[18] P. Kayansamruaj, N. Pirarat, I. Hirono, C. Rodkhum, Increasing of temperature induces

498

pathogenicity of Streptococcus agalactiae and the up–regulation of inflammatory related

499

AC C

497

genes in infected Nile tilapia (Oreochromis niloticus), Vet. Microbiol. 172 (2014) 265–271.

500

[19] J.A. Guijarro, D. Cascales, A.I. García–Torrico, M. García-Domínguez, J. Méndez,

501

Temperature-dependent expression of virulence genes in fish–pathogenic bacteria, Front.

502

Microbiol. 6 (2015) 700–711.

503

[20] S.E. Null, N.R. Mouzon, L.R. Elmore, Dissolved oxygen, stream temperature, and fish

504

habitat response to environmental water purchases, J. Environ. Manage. 197 (2017) 559–570.

505

[21] P. Domenici, J. F. Steffensen, S. Marras, The effect of hypoxia on fish schooling, Philos. T. R.

ACCEPTED MANUSCRIPT 506

Soc. B. 372 (2017) 236–249.

507

[22] T.L. Welker, S.T. Mcnulty, P.H. Klesius, Effect of sub lethal hypoxia on the immune

508

response and susceptibility of channel catfish, Ictalurus punctatus, to enteric septicaemia, J.

509

World Aquac. Soc. 38 (2007) 12–23. [23] M. Abdel-Tawwab, A.E. Hagras, H.A.M. Elbaghdady, M.N. Monier, Effects of dissolved

511

oxygen and fish size on Nile tilapia, Oreochromis niloticus (L.): growth performance,

512

wholebody composition, and innate immunity, Aquacult. Int. 23 (2015) 1261–1274.

513

[24] S.

Gallage,

T.

Katagiri,

M.

Endo,

M.

Maita,

RI PT

510

Comprehensive

evaluation

of

immunomodulation by moderate hypoxia in S. agalactiae vaccinated Nile tilapia, Fish

515

Shellfish Immunol. 66 (2017) 445–454.

SC

514

[25] D. Ndong, Y.Y. Chen, Y.H. Lin, B. Vaseeharan, J.C. Chen, The immune response of tilapia

517

Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low

518

and high temperatures, Fish Shellfish Immunol. 22 (2007) 686–694.

M AN U

516

[26] G.F. Mian, D.T. Godoy, C.A. Leal, T.Y. Yuhara, G.M. Costa, H.C. Figueiredo, Aspects of

520

the natural history and virulence of Streptococcus agalactiae infection in Nile tilapia, Vet.

521

Microbiol. 136 (2009) 180–183.

TE D

519

522

[27] J.L. Zhu, D.Y. Li, Z.Y. Zou, W. Xiao, J. Han, H. Yang, The impact of high temperature stress

523

on serum biochemical parameters and histopathology of Oreochromis niloticus infected by

524

Streptococcus agalactiae, J. Fisheries China 40 (2016) 445–455. [28] W. Li, Y.L. Su, Y.Z. Mai, Y.W. Li, Z.Q. Mo, A.X. Li. Comparative proteome analysis of two

526

Streptococcus agalactiae strains from cultured tilapia with different virulence, Veterinary.

528 529

AC C

527

EP

525

Microbiolog. 170 (2014) 135–143.

[29] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-time quantitative PCR and the 2 (delta delta C (T)) method, Methods 25 (2001) 402–408.

530

[30] K. Hao, J.Y. Liu, F. Ling, X.L. Liu, L. Lu, L. Xia, G.X. Wang, Effects of dietary

531

administration of Shewanella haliotis D4, Bacillus cereus D7 and Aeromonas bivalvium D15,

532

single or combined, on the growth, innate immunity and disease resistance of shrimp,

533

Litopenaeus vannamei, Aquaculture 428–429 (2014) 141–149.

534

[31] J.J. Feng, Y. Lin, S.L. Guo, Y.Y. Jia, Y.L. Wang, F. Zadlock, Z.P. Zhang, Identification and

535

characterization of a novel conserved 46 kD maltoporin of Aeromonas hydrophila as a

ACCEPTED MANUSCRIPT 536

versatile vaccine candidate in European eel (Anguilla anguilla), Fish Shellfish Immunol. 64

537

(2017) 93–103. [32] J. Wang, T. J. Ren, F. Q. Wang, Y. Z. Han, M. L. Liao, Z. Q. Jiang, H. Y. Liu, Effects of

539

Dietary Cadmium on Growth, Antioxidant and Bioaccumulation of Sea Cucumber

540

(Apostichopus japonicus) and Influence of Dietary Vitamin C Supplementation, Ecotox.

541

Environ. Saf. 129 (2016) 145–153.

RI PT

538

[33] X. Ding, D.Q. Lu, Q.H. Hou, S.S. Li, X.C. Liu, Y. Zhang, Orange–spotted grouper

543

(Epinephelus coioides) toll–like receptor 22: Molecular characterization, expression pattern

544

and pertinent signaling pathways, Fish Shellfish Immunol. 33 (2012) 494–503.

SC

542

[34] W. Peng, D.Q. Lu, G.F. Li, X. Zhang, M. Yao, Y. Zhang, H.R. Lin, Two distinct interferon–γ

546

genes in Tetraodon nigroviridis: Functional analysis during Vibrio parahaemolyticus

547

infection, Mol. Immunol. 70 (2016) 34–46.

M AN U

545

[35] F.F. Bai, B. Ni, M.J. Liu, Z.X. Feng, Q.Y. Xiong, S.B. Xiao, G.Q. Shao, Mycoplasma

549

hyopneumoniae-derived lipid-assoiated membrane proteins induce apoptosis in porcine

550

alveolar macrophage via increasing nitric oxide production, oxidative stress, and caspase-3

551

activation, Vet. Immunol. Immunop. 155 (2013) 155–161.

TE D

548

[36] Y.L. Su, J. Feng, C. Liu, W. Li, Y.D. Xie, A.X. Li, Dynamic bacterial colonization and

553

microscopic lesions in multiple organs of tilapia infected with low and high pathogenic

554

Streptococcus agalactiae strains, Aquaculture 471 (2017) 190–203.

555 556

EP

552

[37] K.D. Choi, W. Lehmann, C.A. Harms, J.M. Law, Acute hypoxia–reperfusion triggers immunocompromise in Nile tilapia, J. Aquat. Anim. Health 19 (2007) 128–140. [38] B.O. Kvamme, K. Gadan, F. Finne-Fridell, L. Niklasson, H. Sundh, K. Sundell, Ø. Evensen,

558

Modulation of innate immune responses in Atlantic salmon by chronic hypoxia–induced

559

AC C

557

stress, Fish Shellfish Immunol. 34 (2013) 55–65.

560

[39] F. Buonocore, M. Forlenza, E. Randelli, S. Benedetti, P. Bossù, S. Meloni, C. Secombes, M.

561

Mazzini, G. Scapigliati, Biological activity of sea bass (Dicentrarchus labrax L.)

562

recombinant interleukin-1b, Mar. Biotechnol. 7 (2005) 609–617.

563

[40] J. García-Castillo, E. Chaves-Pozo, P. Olivares, P. Pelegín, J. Meseguer, V. Mulero, The

564

tumor necrosis factor α of the bony fish seabream exhibits the in vivo proinflammatory and

565

proliferative activities of its mammalian counterparts, yet it functions in a species–specific

ACCEPTED MANUSCRIPT 566

manner, Cell. Mol. Life Sci. 61 (2004) 1331–1340.

567

[41] B. Swain, M. Basu, S.S. Lenka, S. Das, P. Jayasankar, M. Samanta, Characterization and

568

inductive expression analysis of interferon gamma-related gene in the India Major Carp,

569

Rohu (Labeo rohita), DNA Cell Biol. 34 (2015) 367–378.

571

[42] M.F. Tsan, B. Gao, Heat shock proteins and immune system, J. Leukoc. Biol. 85 (2009) 905– 910.

RI PT

570

[43] D.C. de Oliveira, F. da Silva Lima, T. Sartori, A.C.A. Santos, M.M. Rogero, R. A. Fock,

573

Glutamine metabolism and its effects on immune response: molecular mechanism and gene

574

expression, Nutrire 41 (2016) 14–24.

SC

572

[44] J. Wang, Y. Wei, X. Li, H. Cao, M. Xu, J. Dai, The identification of heat shock protein genes

576

in goldfish (Carassius auratus) and their expression in a complex environment in Gaobeidian

577

Lake, Beijing, China, Comp. Biochem. Physio. C–Toxicol. Pharmacol. 145 (2007) 350–362.

M AN U

575

578

[45] M. Chen, R. Wang, L. Li, W. Liang, Q. Wang, T. Huang, et al., Immunological enhancement

579

action of endotoxin-free tilapia heat shock protein 70 against Streptococcus iniae, Cell.

580

Immunol. 290 (2014) 1–9.

[46] K.A. Boleza, L.E. Burnett, K.G. Burnett, Hypercapnic hypoxia compromises bactericidal

582

activity of fish anterior kidney cells against opportunistic environmental pathogens, Fish

583

Shellfish Immunol. 11 (2001) 593–610.

TE D

581

[47] A. Castellanos-Gonzalez, L. Jimenez, A. Landa, Cloning, production and characterization of

585

a recombinant Cu/Zn superoxide dismutase from Taenia solium, Int. J. Parasitol. 32 (2002)

586

1175–1182.

EP

584

[48] A.I. Campa-Cordova, N.Y. Hernandez-Saavedra, R. De Philippis, F. Ascencio, Generation of

588

superoxide anion and SOD activity in haemocytes and muscle of American white shrimp

589 590

AC C

587

(Litopenaeus vannamei) as a response to beta-glucan and sulphated polysaccharide, Fish Shellfish Immunol. 12 (2002) 353–366.

591

[49] L.E. Burnett, W.B. Stickle, Physiological responses to hypoxia, in: N.N. Rabalais, R.E.

592

Turner (Eds.), Coastal Hypoxia: Consequences for Living Resources and Ecosystems.

593

Coastal and Estuarine Studies, vol 58, American Geophysical Union, Washington, D.C, 2001,

594

pp. 101–114.

595

[50] J.Y. Wang, J.Y. Wu, L.Y. Yi, Z.X. Hou, W.S. Li, Pathological analysis, detection of antigens,

ACCEPTED MANUSCRIPT 596

FasL expression analysis and leucocytes survival analysis in tilapia (Oreochromis niloticus)

597

after infection with green fluorescent protein labeled Streptococcus agalactiae, Fish Shellfish

598

Immunol. 62 (2017) 86–95.

RI PT

SC

Shellfish Immunol. 9 (1999) 309–318.

M AN U

602

[52] C.M. Press, O. Evensen, The morphology of the immune system in teleost fishes, Fish

TE D

601

101 (2011) 13–30.

EP

600

[51] V.I. Lushchak, Environmentally induced oxidative stress in aquatic animals, Aquat. Toxicol.

AC C

599

ACCEPTED MANUSCRIPT Table 1 Primers used for RT-PCR analysis. Primer sequence

Source

Product (bp)

β-actin-F β-actin-R IL-1β-F IL-1β-R HSP70-F HSP70-R TNF-α-F TNF-α-R IFN-γ-F IFN-γ-R

5΄-TCCATTGGCCTTCGTTGC-3΄ 5΄-CTATTCTGTGTGACCCAGG-3΄ 5΄-ATTGTCGTCCTGTCTATC-3΄ 5΄-AATGTCATCATGGTATTGC-3΄ 5΄-ACCATCACCAACGATAAG-3΄ 5΄-CGGCTTTGTATTTCTCTG-3΄ 5΄-CTGTAGTCACCTCCATTA-3΄ 5΄-TACTTGTTGTTGCTTCTG-3΄ 5΄-CAGCAGAGATGAACTTGA-3΄ 5΄-CACTAGGAAATACGGGTTT-3΄

EF206801

163

GBAY01004231

126

FJ207463

length

RI PT

Primer name

232

121

KF294754

128

SC

GAID01031494

AC C

EP

TE D

M AN U

Abbreviations- IL-1β: interleukin-1beta; HSP70: heat shock protein 70; TNF-α: tumor necrosis factor alpha; IFN-γ: gamma interferon.

ACCEPTED MANUSCRIPT Table 2 Streptococcus agalactiae burden in blood, spleen, head kidney and brain of tilapia at 1 and 3 days (D) post-challenge. Experimental groups

30 °C Blood (CFU/ml) 35 °C

0.92×102 b

0b

4.0 ± 1.0 mg/L DO Con.

10.23×108 a

7.11×105 a

4.0 ± 1.0 mg/L DO Vac.

2.83×102 b

0b

8.0 ± 0.5 mg/L DO Con.

11.36×108 a'

6.48×105 a'

8.0 ± 0.5 mg/L DO Vac.

1.97×102 b'

0 b'

4.0 ± 1.0 mg/L DO Con.

10.41×108 a'

5.64×105 a'

3.38×102 b'

0 b'

5.58×1010 a

5.74×106 a

30 °C

Brain (CFU/g)

35 °C

M AN U

8.0 ± 0.5 mg/L DO Vac.

2.86×103 b

1.12×103 b

4.0 ± 1.0 mg/L DO Con.

8.16×1010 a

9.78×106 a

4.0 ± 1.0 mg/L DO Vac.

1.27×104 c

1.74×104 c

8.0 ± 0.5 mg/L DO Con.

8.24×1010 a'

6.78×106 a'

8.0 ± 0.5 mg/L DO Vac.

2.32×104 b'

2.80×103 b'

4.0 ± 1.0 mg/L DO Con.

9.38×1010 a'

6.47×106 a'

4.0 ± 1.0 mg/L DO Vac.

1.14×105 c'

3.12×104 c'

8.0 ± 0.5 mg/L DO Con.

4.34×109 a

1.56×106 a

8.0 ± 0.5 mg/L DO Vac.

8.96×103 b

1.69×103 b

4.0 ± 1.0 mg/L DO Con.

7.86×108 c

5.40×105 c

4.0 ± 1.0 mg/L DO Vac.

9.20×105 d

2.24×103 b

8.0 ± 0.5 mg/L DO Con.

6.72×107 a'

1.01×106 a'

8.0 ± 0.5 mg/L DO Vac.

1.68×105 b'

2.38×103 b'

4.0 ± 1.0 mg/L DO Con.

1.74×107 a'

1.87×106 a'

4.0 ± 1.0 mg/L DO Vac.

1.12×106 c'

4.76×103 b'

8.0 ± 0.5 mg/L DO Con.

2.27×108 a

3.62×106 a

8.0 ± 0.5 mg/L DO Vac.

4.32×102 b

2.86×103 b

4.0 ± 1.0 mg/L DO Con.

2.40×108 a

3.83×106 a

4.0 ± 1.0 mg/L DO Vac.

1.10×103 c

5.38×103 b

8.0 ± 0.5 mg/L DO Con.

4.31×108 a'

4.54×106 a'

8.0 ± 0.5 mg/L DO Vac.

2.12×103 b'

1.61×104 b'

4.0 ± 1.0 mg/L DO Con.

6.06×108 a'

2.22×106 a'

4.0 ± 1.0 mg/L DO Vac.

9.40×103 b'

3.28×104 b'

EP

AC C

35 °C

3.22×105 a

8.0 ± 0.5 mg/L DO Vac.

TE D

35 °C

Head kidney (CFU/g)

3D 8a

9.10×10

8.0 ± 0.5 mg/L DO Con.

Spleen (CFU/g)

30 °C

1D

8.0 ± 0.5 mg/L DO Con.

4.0 ± 1.0 mg/L DO Vac. 30 °C

Days post challenge

RI PT

Water temperature

SC

Tissue

Notes: Values are given as CFU/ml of blood or CFU/g of tissues, determined by counting bacteria on a BHI plate. At each sampling date, 3 fish were randomly sampled from each group and bacteria counts are the mean of 3 fish. Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (DO = dissolved oxygen; Con. = control; Vac. = vaccinated)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 1. The relative expression of IL-1β (A), TNF-α (B), IFN-γ (C) and HSP70 (D) in spleen of Nile tilapia following vaccination under different oxygen (intermittent hypoxia or normoxia) and rearing water temperature (30 °C or 35 °C) conditions. Bars represent the mean ± SD (n=3). Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (DO = dissolved oxygen; Con. = control; Vac. = vaccinated) IL-1β: interleukin-1beta; TNF-α: tumor necrosis factor alpha; IFN-γ: gamma interferon; HSP70: heat shock protein 70.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 2. The relative expression of IL-1β (A), TNF-α (B), IFN-γ (C) and HSP70 (D) in head kidney of Nile tilapia following vaccination under different oxygen (intermittent hypoxia or normoxia) and rearing water temperature (30 °C or 35 °C) conditions. Bars represent the mean ± SD (n=3). Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (DO = dissolved oxygen; Con. = control; Vac. = vaccinated) IL-1β: interleukin-1beta; TNF-α: tumor necrosis factor alpha; IFN-γ: gamma interferon; HSP70: heat shock protein 70.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 3. (A) superoxide dismutase (SOD) activity, (B) catalase (CAT) activity, (C) glutathione peroxidase (GSH-Px) activity and (D) malondialdehyde (MDA) levels of Nile tilapia following vaccination under different oxygen (intermittent hypoxia or normoxia) and rearing water temperature (30 °C or 35 °C) conditions. Bars represent the mean ± SD (n=3). Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (DO = dissolved oxygen; Con. = control; Vac. = vaccinated)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 4. The phagocytic capacity of PBLs was detected under different oxygen (intermittent hypoxia or normoxia) and rearing water temperature (30 °C or 35 °C) conditions by FCM at 28 d post-vaccination. (A) PBLs with FITC-bacteria were gated (P) on forward and side scatter (FS-SS) do plot, Q demonstrated the bacteria distribution (no showed); (B) Phagocytic percentage were statistically calculated. Histograms showed the phagocytic percentage. Bars represent the mean ± SD (n=3). Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (No. = normoxia; In. Hy. = intermittent hypoxia; Con. = control; Vac. = vaccinated)

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 5. Respiratory burst activity of Nile tilapia primary spleen cells at 30°C (A) or 35°C (B) and primary head kidney cells at 30°C (C) or 35°C (D) after stimulation with NBT, S. agalactiae and PMA was detected at 28 d post-vaccination. Bars represent the mean ± SD (n=3). Different superscript letters (a, b, c and d) indicate significant differences (p < 0.05). (No. = normoxia; In. Hy. = intermittent hypoxia; Con. = control; Vac. = vaccinated)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 6. Nitric oxide production of Nile tilapia primary spleen cells (A) and primary head kidney cells (B) after stimulation with S. agalactiae was detected at 28 d post-vaccination. Bars represent the mean ± SD (n=3). Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (DO = dissolved oxygen; Con. = control; Vac. = vaccinated)

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Fig. 7. Histomorphology of distal intestine (In) and head kidney (HK) in vaccinated Nile tilapia under different oxygen (intermittent hypoxia or normoxia) and rearing water temperature (30 °C or 35 °C) at 28 d post-vaccination. In-A: 30 °C and normoxia; In-B: 30 °C and intermittent hypoxia; In-C: 35 °C and normoxia; In-D: 35 °C and intermittent hypoxia; HK-A: 30 °C and normoxia; HK-B: 30 °C and intermittent hypoxia; HK-C: 35 °C and normoxia; HK-D: 35 °C and intermittent hypoxia. Thickness 4 µm. In bar = 100 µm, HK bar = 50 µm.

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Fig. 8. Cumulative mortality (%) of tilapia was recorded daily for 14 d post intraperitoneal challenge with S. agalactiae THN0901 (2×107 CFU/fish). Tilapia mortalities occurred in large quantities during the first week of the challenge. 384 fish were used with 48 per group. Each value represents mean (n=3) and error bars are omitted for clarity. Different superscript letters indicate significant differences (p < 0.05). a/b/c/d denotes the difference among the groups at 30 °C. a'/b'/c'/d' denotes the difference among the groups at 35 °C. (No. = normoxia; In. Hy. = intermittent hypoxia; Con. = control; Vac. = vaccinated)

ACCEPTED MANUSCRIPT

Highlights: Intermittent hypoxia at either 30°C or 35°C modulates immune genes expression. Enzymatic activities were lower in vaccinated hypoxia fish at both temperatures. Phagocytosis and ROS production decreased in vaccinated hypoxia fish.

RI PT

Cumulative mortality was higher in vaccinated hypoxia fish. Spleen, head kidney and brain bacteria burden was lower in vaccinated normoxic

AC C

EP

TE D

M AN U

SC

fish.