For Review Only › sjstweb › Ar-Press › 2020May › 31.pdf · Sheikh, Hassan; Universiti Malaysia Terengganu, Faculty of fisheries and food Science Abdul Wahid, Mohd Effendy;

For Review OnlyPotential of Chlorella sp. exopolysaccharide as adjuvant for

Mannheimia haemolytica A2 vaccine in rat model.

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2019-0494.R1

Manuscript Type: Original Article

Date Submitted by the Author: 20-Apr-2020

Complete List of Authors: Kamaradin, Insyirah-husna; Universiti Malaysia Terengganu, Institute of Marine BiotechnologyAssaw, Suvik; Universiti Malaysia Terengganu, Faculty of Marine Science and Environment; Universiti Sains Malaysia, Department of biomedical science, School of Health ScienceSheikh, Hassan; Universiti Malaysia Terengganu, Faculty of fisheries and food ScienceAbdul Wahid, Mohd Effendy; Universiti Malaysia Terengganu, Faculty of Fisheries and Food Science; Universiti Malaysia Terengganu, Institute of Marine Biotechnology

Keyword: Exopolysaccharide, Mannheimia haemolytica A2, adjuvant, immunoglobulins, intranasal.

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2019-0494.R1 Sheikh

For Review Only

Manuscript ID SJST-2019-0494 entitled "Potential of Chlorella sp. exopolysaccharide as adjuvant for Mannheimia haemolytica A2 vaccine in rat model."

Comment Response

The degree of increased/decreased (how many times) for each parameters measured are describe in the result part.

We appreciate this comment, however, the increases were significant but not as high as 2 or 3 folds. Here are some examples:

1- IgM titers in EPS-MhA2 treated group starting at week 5 till week 6 increase significantly (about 20%) but wasn’t as high as 2 folds for example.

2- EPS-MhA2 vaccine also significantly increased IgA titer after booster dose at week 5 (about 67%), which is also less than 2 folds.

The levels of the antibodies were expressed as the absorbance unit (Fig 7). Is it possible to show the antibody level in concentration unit? Since the first antibody normally produced during a primary immune response is Ig M. However, the absorbance of Ig G were higher than IgM during wk 0 and wk 1 in both MHA2 and EPS-MHA2 groups.

We didn’t convert the units because first, the data were sufficient to show the effect of vaccination. Secondly, we will need large number of animals to establish a cut-off point which is not practical at this point of the research.

The starting point of IgG was already higher than IgM, because of 2 main reasons. First, IgG is in the serum, while IgA circulates and enter the bloodstream with the secretion of localized BALT in the lungs. Secondly, the animals used are not specific-pathogen free (SPF) animals.

Gough, N. R. (2006) reported that antibody-producing cells undergo a process of differentiation and class switch recombination (CSR) such that the antibodies produced start as immunoglobulin M (IgM) and then switch to IgG and IgA as the concentration of antigen changes and as the cells differentiate. Therefore, the initial antibody levels circulating in the serum were not significantly important since they are not specific to the antigen introduced during the first exposure, with the a condition that it does not exceed twice of the control animal antibody’s level.

Page 1 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

At wk 1 post vaccination (Fig 7), all groups showed gradual increase in Ig A (p > 0.05), except PBS ?? (the original text was MHA2

The correct word is “PBS” and the text has been edited.

The role of Ig A for local immunity should be of concern. Would it be possible to address whether Ig A can be detected in the secretion of upper respiratory tract region ?

Even though the upper respiratory tract is suitable specially to study NALT (nasal-associated respiratory tract), however, the selected pathogen usually colonizes and invade the lower respiratory tract (bronchioles and lungs). In the nasal there is air turbinates that would be difficult to process for histology examination.

Figures : Minor editing on figure legends Figures number corrected

Reference:

Gough, N. R. (2006). How to Get from IgM to IgG. Science Signaling, 2006(358). doi: 10.1126/stke.3582006tw365.

Page 2 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

1 Title: Potential of Chlorella sp. exopolysaccharide as adjuvant for Mannheimia

2 haemolytica A2 vaccine in rat model.

3

4 Insyirah-husna Kamaradin3, Suvik Assaw2,3, Hassan, I. Sheikh1 and Mohd Effendy

5 Abd Wahid1,3*

6

7 1Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, Terengganu,

8 Malaysia;

9 2Faculty of Marine Science and Environment, Universiti Malaysia Terengganu,

10 Terengganu, Malaysia

11 3Institute of Marine Biotechnology, Universiti Malaysia Terengganu, Terengganu,

12 Malaysia;

13 4Department of Biomedical Sciences, School of Health Science, Unversiti Sains

14 Malaysia, 16150 Kota Bharu, Kelantan.

15

16

17 *Corresponding Author: [email protected]

18

19

20

21

22

23

24

25

26

27

28

29

30

31 ABSTRACT

Page 3 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

32 The potential of exopolysaccharides (EPS) extracted from Chlorella sp. as adjuvant for

33 formalin-killed whole organism of Mannheimia haemolytica A2 (MhA2) vaccine was

34 investigated. Chlorella sp. was cultured in F/2 media and EPS was extracted and

35 characterized using GPC, FTIR and SEM, while its cytotoxicity was tested on RAW

36 264.7. Sprague Dawley rats were inoculated intranasally with either phosphate-buffered

37 saline (PBS) (Group A), EPS only (Group B), formalin killed whole organism of MhA2

38 vaccine seed only (Group C) or EPS-MhA2 adjuvanted vaccine (Group D). Serum levels

39 of IgM, IgG, and IgA titers were collected for nine weeks. About 4.8 mg of EPS was

40 obtained in the form of white powder with molecular weight of 2398 Da. The

41 microstructure of EPS was compact and exhibited flakes like structural units and was not

42 toxic to RAW 264.7. EPS-MhA2 group produced significantly higher (p<0.05) IgM, IgG

43 and IgA responses compared to other groups.

44

45 Keywords: Exopolysaccharide, Mannheimia haemolytica A2, adjuvant,

46 immunoglobulins, intranasal.

47

48 INTRODUCTION

49 Vaccination is the most effective strategy in the prevention of infectious diseases by

50 generating immune responses and maintain memory cells that able to induce rapid recall

51 responses (Bartlett & Tyring, 2009; Sarkander, Hojyo, & Tokoyoda, 2016). The entry

52 point of more than 90% of pathogens is via mucosal membrane and mucosal vaccination

53 is an effective approach (Miquel‐Clopés, Bentley, Stewart, & Carding, 2019). The

54 vascularized mucosal surface area is 150 cm3 at the nasopharyngeal compartment and can

Page 4 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

55 be targeted for vaccine uptake (Holmgren & Czerkinsky, 2005; Olszewska & Steward,

56 2001; Wang, Liu, Zhang, & Qian, 2015).

57 Pneumonic mannheimiosis caused by Mannheimia haemolytica serotype A2, a gram-

58 negative commensal bacterium that can be found at the upper respiratory tract of domestic

59 and wild animals including healthy ruminants (Roier et al., 2013). The bacterium can

60 turn opportunistic when the animals are exposed to predisposing factors such as

61 pregnancy, transportation or introduction of new animals into an existing herd.

62 Pneumonic mannheimiosis can lead to high mortality and economic losses to the ruminant

63 industry (Jesse et al., 2014).

64 M. haemolytica initiates their infection at the lining mucosal surfaces of the lower

65 respiratory system (Wang et al., 2015). The efficacy of mucosal vaccination through an

66 intranasal route of delivery had been successful in small ruminants (Effendy, Zamri-Saad,

67 Puspa, & Rosiah, 1998a; Effendy, Zamri-Saad, Mohamad, & Omar, 1998b; Zamri-Saad,

68 Effendy, Israf, & Azmi, 1999a; Zamri-Saad, Maswati, Effendy, & Jasni, 1999b).

69 However, the uptake of vaccines from the nasal cavity is poor due to rapid clearance and

70 poor transportation across the nasal mucosal membrane (Svindland et al., 2012).

71 Therefore, the intranasal administration of formalin-killed whole organism of M.

72 haemolytica A2 (MhA2) vaccine would need an extra component, such as an adjuvant

73 that is capable of sustaining their presence and ameliorating the immune responses.

74 Exopolysaccharide (EPS) is an organic macromolecule that consist of various types of

75 sugars and is produced by many microorganisms. The EPS can be found either as capsular

76 matrix that bound covalently to the cell surface or as slime materials that bound loosely

77 (Ahmed, Wang, Anjum, Ahmad, & Khan, 2013; Badel, Bernardi, & Michaud, 2011). In

78 marine environment, EPS form biofilm, which allows the cells to adhere to other bacteria,

Page 5 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

79 animal, plant tissues or inert surfaces (Sutherland, 2001). Due to these properties, EPS

80 could have muco-adhesives character that might be suitable as mucosal adjuvant that

81 could ensure long retention and high absorption rates in the nasal cavity (Svindland et al.,

82 2012; Zaman, Chandrudu, & Toth, 2013).

83 In recent studies, EPS from marine origin such as microalgae has been used in

84 pharmaceuticals industry due to its immune-modulatory, apoptotic and antiviral activity

85 (Xiao & Zheng, 2016; Ishiguro et al., 2017). Hence, EPS from Chlorella sp. was chosen

86 to elucidate its potential as a mucosal adjuvant vaccine for formalin-killed whole

87 organism of Mannheimia haemolytica A2 vaccine. The study aims at evaluating the in

88 vivo adjuvant activity of EPS using Sprague Dawley rat model to determine whether EPS

89 can enhance the humoral immunity in systemic compartments by examining the

90 development of antibodies (IgM, IgG and IgA) responses.

91

92 MATERIALS AND METHOD

93 Extraction of EPS from Chlorella sp.

94 A pure culture of Chlorella sp. (1x106 CFU/mL) that was cultured into F/2 media at ratio

95 of 1:1, was centrifuged at 15,000 rpm for 20 minutes at 4oC using refrigerated centrifuge

96 in order to extract EPS following previously established method with slight modifications

97 (Bajpai, Majumder, Rather, & Kim, 2016). Then, supernatant produced were mixed with

98 100% ice-cold ethanol at ratio 1:1 (25 mL/ 25 mL) and incubated at 4oC overnight. After

99 24 hours of incubation, the solution was centrifuged again at 15,000 rpm for 20 minutes

100 at 4oC. The pellet was collected and washed twice with distilled water then dissolved in

101 distilled water. The pellet was then dialyzed using 16.5 x 26 mm visking tubing

102 membrane for 24 hours at 4oC to remove protein residues. Next, the solution in visking

Page 6 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

103 tube was transferred into the falcon tube and freeze-dried using vacuum freeze dryer. The

104 dried pellet obtained was weighted and stored refrigerated at -20oC.

105 Identification of EPS from Chlorella sp.

106 Gel Permeation Chromatography (GPC)

107 The molecular weight of EPS pellet was analyzed using Gel Permeation Chromatography

108 (GPC) method using a Waters 1515 HPLC system equipped with Waters 2414 refractive

109 index detector (RID). Three types of columns, namely 7.8 x 300 mm UltrahydrogelTM

110 500 columns, 7.8 x 300 mm UltrahydrogelTM 120 columns and 7.8 x 300 mm

111 UltrahydrogelTM Linear columns were used. Then, 1 mg of EPS was dissolved in 1mL of

112 0.1 M NaNO3 and injected. The eluent was 0.1 M NaNO3 in water. The columns and

113 detector were maintained at 35oC. The sample was analyzed within 40 minutes at a flow

114 rate of 1 mL/min and the injection volume was set at 25 μL. Dextran Molecular Weight

115 Standard Kits (Polymers Standards Service-USA, Inc. Germany) was used in the GPC as

116 a standard. The data collection and analysis were carried out using a Breeze 2 software

117 (Xing et al., 2017).

118

119 Fourier Transform Infrared spectroscopy (FTIR)

120 The spectra of EPS were recorded using spectrophotometer (Perkin Elmer, USA) using

121 Potassium Bromide (KBr) disc. FTIR absorption band were read at wavelengths (λ)

122 between 2.5 μm to 25 μm (1 μm = 10-6 m) and measured in cm-1 unit (Siti-Aisha, 2018).

123

124 Scanning Electron Microscope (SEM)

125 The diluted EPS sample (106 CFU/mL) was centrifuged at 4,000 rpm for 15 minutes. The

126 pellet obtained was placed on a small circle glass slide and dried in room temperature for

Page 7 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

127 15 minutes. Next, the slide with dried pellet was placed on stubs, then coated with the

128 gold 20-40 μm thicknesses using auto fine coated JFC 1600 and viewed under SEM (Akin

129 & Amos, 1975).

130

131 Cytotoxicity test of EPS

132 Measurement of cell viability assay by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-Diphenyl-

133 Tetrazolium Bromide (MTT)

134 Viability of the cell against EPS was determined via 3-(4,5-dimethylthiazol-2-yl)-2, 5-

135 Diphenyl-Tetrazolium Bromide (MTT) assay (Razali et al., 2014). Briefly, RAW 264.7

136 cells that obtained from the American Type Culture Collection (ATCC) was cultured

137 according to the method described by Kim et al. (2015). After the cells culture reached

138 80% confluence in the culture flask, cells were seeded in 96 wells plate at the density of

139 5 x 104 cells/well. After 24 hours of incubation, the adhered cells were treated with various

140 concentrations of the EPS that was diluted in DMEM (100 µg/mL, 50 µg/mL, 25 µg/mL,

141 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL, and 1.56 µg/mL, Control positive (DMEM only).

142 Twenty-four hours later, MTT was added and the cells were incubated for 4 hours at 37°C

143 and 5% CO2. The medium was then removed, and the formazan precipitate was

144 solubilized in DMSO. The absorbance was measured at 550 nm by using microplate

145 reader (Thermo-Scientific Multiskan AscentTM, USA) for cell viability calculation. The

146 cytotoxicity of EPS was expressed as the IC50, which is the concentration of each EPS

147 that reduces the 50% of cell survival when compared with the control.

148 Cell viability (%) = (OD550 of sample) (OD550 of control)-1 x 100

149

150

Page 8 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

151 Preparation of formalin-killed cells (FKCs) of Mannheimia haemolytica A2 (MhA2)

152 vaccine

153 Mannheimia haemolytica A2 were cultured on Thermo Scientific Columbia Sheep Blood

154 Agar (BA) plate. About 30 colonies of the pure bacteria were transferred into 200 mL of

155 Brain Heart Infusion broth. The culture was then shook by using shaker incubator at 37°C

156 for 18 hours for replications. Following incubation, serial dilutions and standard plate

157 counting were done to determine the concentration of the culture. The bacteria were then

158 killed by introducing neutral-buffered formalin at the concentration of 1% (0.25 mL/ 25

159 mL) formalin in phosphate buffered saline and kept overnight at 4°C. Then the bacteria

160 were washed five times with PBS by centrifugation at 6000 xg for 15 minutes using

161 refrigerated centrifuge to eliminate the remaining formalin from the cultures. The bacteria

162 were washed again five times with sterile PBS, and the concentration in preparation was

163 adjusted to 106 CFU/mL using optical density (Noraini, Sabri, & Siti-Zahrah., 2013).

164

165 Preparation of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)

166 vaccine

167 The 4.8 mg of EPS powder were diluted in 400 mL of phosphate buffered saline to prepare

168 as a stock solution with a concentration 0.012 mg/mL. Then only 30 μL of diluted EPS

169 was taken and mixed together with the pellet of 106 CFU/mL of formalin-killed M.

170 haemolytica A2 (MhA2) vaccine to make exopolysaccharide-M. haemolytica A2 (EPS-

171 MhA2) vaccine (Noor-Hidayah, 2014).

172

173

Page 9 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

174 Confirmation of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)

175 vaccine

176 Scanning Electron Microscope (SEM)

177 The mixture of diluted EPS and 106 CFU/mL of MhA2 vaccines were centrifuged at 4,000

178 rpm for 15 minutes using centrifuge. Then, the pellet was collected and placed on small

179 round glass slide and dried in room temperature for 15 minutes. The slide with dried pellet

180 was coated with the gold 20-40 μm thicknesses using auto fine coated JFC 1600 and ready

181 to view in low vacuum condition under SEM (Akin and Amos, 1975).

182

183 Experimental design of white rats (Sprague Dawley)

184 Twelve female healthy Sprague Dawley of 6-8 weeks age were selected and allocated

185 randomly into 4 groups with 3 rats each group assigned as group A, group B, group C

186 and group D. Group A and B were negative control treated with phosphate buffered saline

187 and EPS as adjuvant respectively. Group C was positive control treated with MhA2

188 vaccine only and Group D was treated with EPS-MhA2 vaccine, which served as

189 adjuvanted vaccine. All Sprague Dawley were inoculated by dripping 30 µL of samples

190 via the intranasal route. Blood samples were taken every week from each of the Sprague

191 Dawley’s tail vein for determining the antibody production of the rats. The first treatments

192 were administered into the Sprague Dawley’s nostril and labeled as week 0. Then on week

193 2, second booster dose was given. Six weeks after 2nd vaccination (week 8), all rats were

194 euthanized according to the animal ethics code UMT/JKEPHT/2017/2.

195

196

197

Page 10 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

198 RESULTS

199 Physical and chemical characterization of exopolysaccharide (EPS) from Chlorella

200 sp.

201 The 400 mL of Chlorella sp. culture containing 106 CFU/mL had yielded 4.8 mg in the

202 form of white powder that and was readily soluble in water (Figure 1). Gel Permeation

203 Chromatography (GPC) revealed that the average molecular weight (Mw) of the EPS was

204 2398 Da as shown in (Figure 2). FTIR spectrum indicates the major functional groups

205 and chemical bonds present in EPS. The peak at 3350.35cm-1 indicated O-H stretch. A

206 weak absorption band at the wave-number region <3000 cm-1 was assigned to the

207 symmetric stretching vibration of CH group. A sharp peak found at 1683.86 cm-1 and

208 1402.25 cm-1 indicated stretching vibrations of C=O and C-O respectively which are

209 linked to COOH groups. While the absorption bands in the region 1116.78 cm-1 and

210 975.98cm-1 suggested a typical signature of carbohydrates molecules (Figure 3).

211 Morphology of freeze-dried EPS was visualized by Scanning Electron microscope (SEM)

212 analysis at 2500x magnification. The microstructure of freeze-dried EPS was found to

213 have compact in structure and flakes like structural unit structure with sharp edges (Figure

214 4).

215

216 Cytotoxicity of exopolysaccharide (EPS) from Chlorella sp.

217 The in vitro cytotoxicity test of EPS was evaluated using MTT assay on Mouse

218 Leukaemic monocyte-macrophages cell line (RAW 264.7). The MTT assay was

219 performed as percentage relative viability of cells (%) versus concentrations of EPS

220 extracts (100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL, and

221 1.56 µg/mL) and the percentage viability of RAW 264.7 cell was calculated after 24 hours

Page 11 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

222 exposure in concentration-dependent manner. The results as shown in Figure 5 indicated

223 that EPS promoted the growth of RAW 264.7 cell at all concentrations (3.125 µg/mL

224 (112.21%), 6.25 µg/mL (125.07%), 12.5 µg/mL (140.34%), 25 µg/mL (143.97%), 50

225 µg/mL (152.01%), 100 µg/mL (140.84%)) except at this concentrations 1.56 µg/mL

226 (67.24%) of EPS where the RAW 264.7 growth was lower than control (100%). There

227 was no significant differences (p>0.05) in percentage relative viability of cell between

228 tested concentrations and the control. No IC50 value was obtained, hence, EPS is safe at

229 all tested concentrations.

230

231 Morphology of exopolysaccharide-Mannheimia haemolytica A2 (EPS-MhA2)

232 vaccine

233 In order to confirm whether the EPS powder able to encapsulate whole-cell formalin

234 killed M. haemolytica A2, the morphological structure of the EPS-MhA2 was observed

235 under SEM. Based on Figure 6, the clumping structure showed the EPS had successfully

236 encapsulated the whole-cell formalin-killed M. haemolytica A2 under 6,1500x

237 magnification.

238

239 Development pattern of IgM, IgA and IgG of white rats (Sprague Dawley)

240 Enzyme-linked Immunosorbent Assay (ELISA) results revealed that there were three

241 types of response patterns showed by IgM, IgG and IgA antibodies in the blood serum of

242 experimental Sprague Dawley rats before and after vaccination (Figure 7). At the starting

243 week (week 0) before vaccination program, the levels of IgM, IgG and IgA in all Sprague

244 Dawley were at low with no significant (p>0.05) differences between groups. However,

Page 12 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

245 following the 1st intranasal vaccination on week 1, the development of IgM, IgG and IgA

246 responses in all groups of Sprague Dawley showed a different pattern of responses.

247 For the IgM, after the 1st intranasal vaccination on week 1, the level of IgM in all groups

248 showed a gradual increase but the increment was not significant (p>0.05) until week 3.

249 However, following a booster dose on week 2, despite slight decrease at week 3, both

250 EPS-MhA2 and M. MhA2 vaccinated groups showed significant (p<0.05) increase at

251 week 4 when compared with both unvaccinated groups. At week 5 and week 6, only EPS-

252 MhA2 group sustained a significant increase (p<0.05) when compared to other groups.

253 For IgG, at week-1 post intranasal vaccination, the mean antibody levels of IgG showed

254 significant (p<0.05) increase in EPS-MhA2 and M. MhA2, while insignificant (p>0.05)

255 gradual increase was observed in EPS-only and PBS groups before the mean antibody

256 level started to decline at week 2 and 3 post-vaccination. After that, the 2nd intranasal

257 vaccination of EPS-MhA2 and MhA2 vaccine group showed that it had successfully

258 induced IgG production. However, EPS-MhA2 vaccine group produced highest and

259 significant (p<0.05) increase when compared with MhA2 vaccine group and the

260 unvaccinated control groups at week 5. Meanwhile for IgA, similar pattern with IgG was

261 observed. At week 1 post intranasal vaccination all groups except PBS showed gradual

262 increase in antibody, but the levels were not significant (p>0.05). The booster dose

263 showed again that EPS-MhA2 and MhA2 vaccine group significantly (p<0.05) increased

264 the antibody levels of IgA at week 5 as compared to the control groups. Among

265 vaccinated groups, IgA in EPS-MhA2 vaccine group was still significantly (p<0.05)

266 higher than MhA2 vaccine group.

267 Overall, from the development pattern of IgM, IgG and IgA, only EPS-MhA2 group

268 consistently demonstrated a significant increased (p<0.05) in the production of IgM, IgG

Page 13 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

269 and IgA, especially after administration of booster doses at two weeks interval. The level

270 of IgM started to increase after the 2nd booster on week 4 and peaked at week 6, while

271 IgG and IgA reach their peak at week 5, respectively when compared with the rest of the

272 non-adjuvanted groups.

273

274 DISCUSSION

275 Chlorella sp. is known to produce and excrete large amounts of EPS in their extracellular

276 environment (Delattre, Pierre, Laroche, & Michaud, 2016). Chlorella sp. culture medium

277 was used to extract EPS. The yield EPS obtained in this study from Chlorella sp. culture

278 using 106 CFU/mL was about 120 mg/L. In another study, yield of EPS obtained from

279 Chlorella sp. was 174 mg/L (Xiao & Zheng, 2016), however, the study didn’t report the

280 concentration of the Chlorella sp. Physiochemical, structural, and biological activity of

281 the EPS were determined to assess their biodegradability and toxicity to be used as

282 vaccine adjuvant (Ahmed et al., 2013). EPS produced in this study was in the form of

283 white powder and was highly water soluble. The EPS showed a single symmetrical peak

284 in Gel Permeation Chromatography (GPC) profile, which indicated that the extracted EPS

285 was a homogeneous polysaccharide (Du, Yang, Bian, & Xu, 2017). The average

286 molecular weight (Mw) of purified EPS was 2398 Da, which was relatively small in size

287 and categorized as low molecular weight. Xiao and Zheng (2016) suggested that EPS

288 from Chlorella sp. should have high molecular weight due to its structural

289 polysaccharides, which mainly consist of 1-6 galactose linkage. Noor-Hidayah (2014)

290 also reported low molecular weight of Chlorella sp. to be as low as 2274 Da. The low

291 molecular weight EPS could be due to degradation of polysaccharide chain (Ziadi et al.,

292 2018) or due to limited number of monosaccharides present such as glucose, xylose,

Page 14 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

293 glucuronic acid and galactose (Raposo, de Morais, and de Morais, 2015; Xiao & Zheng,

294 2016). However, low molecular weight is advantageous as it associated with higher water

295 solubility, stability and organism absorption (Ramnani et al., 2012), hence, suitable to be

296 used as adjuvant vaccines. Fourier-transform infrared spectroscopy (FTIR) spectra of

297 Chlorella sp. EPS was in agreement with previous studies (Bayona & Garcés, 2014; Sajna

298 et al., 2013; Wang, 2011). The OH group at 3350.35 cm-1 could allow EPS to be absorbed

299 easily by the mucus of nasal cavity and increase the nasal residence time for the vaccine

300 (Sahoo, Chakraborti, & Mishra, 2011; Yang, Zhao, & Fang, 2008; Zaman et al., 2013).

301 SEM properties of Chlorella sp.’s EPS were similar to the properties of EPS reported in

302 previous studies (Ahmed et al., 2013; Du et al., 2017; Piermaria, Pinotti, Garcia, &

303 Abraham, 2009) where the surface of EPS was dull and had pores. The microstructure of

304 the produced EPS was similar to materials used to make plasticized films which indicated

305 high water solubility, water absorbency and biodegradability. The EPS was also

306 homogeneous matrix, which indicated the structural integrity and ability to make a film

307 (Ahmed et al., 2013). The morphology of EPS changed when it was mixed with sterile

308 PBS excipient and the seed of formalin-killed whole organism MhA2 (Figure 6).

309 Variations in EPS structure is common due to differences in physicochemical properties

310 of the polysaccharide itself (Kanamarlapudi & Muddada, 2017). Chlorella sp.’s EPS did

311 not show any cytotoxic effect on RAW 264.7 macrophage cell by MTT assay at all the

312 tested concentrations between 1.56 to 100 µg/ml. This finding is consistent with previous

313 study where the polysaccharide extracted from green microalgae Haematococcus

314 lacustris on RAW 264.7 macrophage cells had immune-modulating activity without any

315 detectable level of cytotoxicity (Park et al., 2011).

Page 15 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

316 Chlorella sp. EPS potential to be used as an adjuvant for M. haemolytica A2 (MhA2)

317 vaccine at mucosal surfaces. The development patterns of the antibody in the blood serum

318 are direct indicator of the humoral immune responses and generally used for examining

319 the effectiveness of vaccine (Yang et al., 2015). In this study, IgM, IgG and IgA

320 production was monitor in the blood serum of Sprague Dawley (Figure 7). Generally,

321 EPS-MhA2 adjuvanted vaccine group showed higher level of antibodies, especially after

322 2nd intranasal vaccination at week 2. Significant increase in IgM titers in EPS-MhA2

323 treated group starting at week 4 till week 6 could be due to opsonizing effect of serum

324 IgM towards the exposure of EPS-MhA2. This result was supported by the fact that the

325 IgM would be secreted out into the blood as the first antibody production to take action

326 during the primary and secondary responses of any antigen (Black & Black, 2008). Then,

327 the antibody response was followed by IgG production. In this study, there was increase

328 of IgG level in Sprague Dawley in the two vaccinated groups (EPS-MhA2 and MhA2).

329 The increase of IgG level from these groups was gradually stimulated starting at week 4

330 and then became significantly high at week 5. However, EPS-MhA2 vaccine showed

331 significantly higher IgG responses compared to MhA2 only vaccine. This observation

332 indicated that the EPS as adjuvant had a good ability to maintain the M. haemolytica A2

333 vaccine in mucosal region of the Sprague Dawley, thus gave appropriate time for this

334 antigens to stimulate memory cells during primary response. Due to the adequate

335 production of these memory cells, the administration of the booster dose invigorated the

336 significantly high level of IgG on systemic immune compartment. This finding was

337 similar to the previous study where EPS adjuvant from Aphanothece halophytica

338 (EPSAH) significantly enhanced serum OVA-specific total IgG and IgG2a production in

339 immunized mice at 2 weeks post-immunization (Lei et al., 2016). EPS-MhA2 vaccine

Page 16 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

340 also significantly increased IgA titer in the blood serum of Sprague Dawley at week 5,

341 especially after booster dose (week 2) in EPS-MhA2 group compared to the other groups

342 before the production of IgA declined on week 6. The increase in IgA production in this

343 study was consistent with the observations of IgA responses on mice that were vaccinated

344 with the chitosan polysaccharide-adjuvanted vaccine (Svindland et al., 2012).

345 The findings from this study suggest that the mechanism of the adjuvant is that the

346 microencapsulation of the seeds will protect the seed from being phagocytized by the

347 cells of the innate immune system. Neutrophils will engulf the adjuvant and degrade them,

348 eventually exposing the seeds. Macrophages will clear the remaining EPS capsules and

349 after a few hours, the process continues with the bindings of B cells receptors with the

350 naked antigen seeds before the proliferation or cloning process of antibodies took place

351 (Sompayrac, 2019). The receptors will bind directly to the antigens and continue the

352 cloning process to produce huge quantities of specific antibodies into the tissues and

353 blood. Instead of two weeks interval, it is better to give the booster at three weeks interval

354 due to get more impactful immune responses due to the delay from the innate immune

355 system to remove the EPS.

356

357 CONCLUSION

358 EPS from Chlorella sp. was found to possess suitable physiochemical properties to be a

359 good adjuvant for intranasal delivery. EPS from Chlorella sp. significantly increase of

360 IgM, IgG and IgA humoral immune responses and hence an effective adjuvant for M.

361 haemolytica A2 vaccine. EPS as intranasal adjuvant enablabled M. haemolytica A2

362 vaccine seed to stay longer on the nasal mucosal epithelium. This will aid antigens to get

363 better access to the lymphoid tissue and thereby stimulate an appropriate immune

Page 17 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

364 response in preventing the establishment of this particular infection. EPS obtained from

365 Chlorella sp. could also be used as an adjuvant in other vaccines, especially those

366 administered through intranasal or mucosal areas.

367

368 ACKNOWLEDGEMENT

369 The study was funded by the Fundamental Research Grant Scheme (FRGS) (VOT

370 59338) from the Ministry of higher Education

371

372 REFERENCES

373 Ahmed, Z., Wang, Y., Anjum, N., Ahmad, A., & Khan, S. T. (2013). Characterization of

374 exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from

375 Tibet kefir–Part II. Food Hydrocolloids, 30(1), 343-350.

376 Akin, D. E., & Amos, H. E. (1975). Rumen bacterial degradation of forage cell walls

377 investigated by electron microscopy. Appl. Environ. Microbiol., 29(5), 692-701.

378 Badel, S., Bernardi, T., & Michaud, P. (2011). New perspectives for Lactobacilli

379 exopolysaccharides. Biotechnology advances, 29(1), 54-66.

380 Bajpai, V. K., Majumder, R., Rather, I. A., & Kim, K. (2016). Extraction, isolation and

381 purification of exopolysaccharide from lactic acid bacteria using ethanol

382 precipitation method. Bangladesh Journal of Pharmacology, 11(3), 573-576.

383 Bartlett, B. L., & Tyring, S. K. (2009). Safety and efficacy of vaccines. Dermatologic

384 Therapy, 22(2), 97-103. doi: 10.1111/j.1529-8019.2009.01222.x

385 Bayona, K. C. D., & Garcés, L. A. (2014). Effect of different media on exopolysaccharide

386 and biomass production by the green microalga Botryococcus braunii. Journal of

387 applied phycology, 26(5), 2087-2095.

388 Black, J. G., Black, L. J. . (2008). Microbiology: Principles and explorations. (7 ed.).

389 Hoboken, NJ: John Wiley & Sons, Inc.

390 Delattre, C., Pierre, G., Laroche, C., & Michaud, P. (2016). Production, extraction and

391 characterization of microalgal and cyanobacterial exopolysaccharides.

392 Biotechnology advances, 34(7), 1159-1179.

Page 18 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

393 Du, B., Yang, Y., Bian, Z., & Xu, B. (2017). Characterization and anti-Inflammatory

394 potential of an exopolysaccharide from submerged mycelial culture of

395 Schizophyllum commune. Frontiers in pharmacology, 8, 252.

396 Effendy, A., Zamri-Saad, M., Puspa, R., & Rosiah, S. (1998a). Efficacy of intranasal

397 administration of formalin-killed Pasteurella haemolytica A2 against

398 intratracheal challenge in goats. Veterinary record, 142(16), 428-431.

399 Effendy, A. W. M., Zamri-Saad, M., Mohamad, M. and Omar, H. . (1998b). Vaccination

400 of sheep against pneumonic pasteurellosis using a new spray vaccine. Jurnal

401 Veterinar Malaysia, 10, 17-20.

402 Holmgren, J., & Czerkinsky, C. (2005). Mucosal immunity and vaccines. Nature

403 medicine, 11(4), 45-53.

404 Ishiguro, S., Uppalapati, D., Goldsmith, Z., Robertson, D., Hodge, J., Holt, H.,

405 Nakashima, A., Turner, K., and Tamura, M. (2017). Exopolysaccharide

406 extracted from Parachlorella kessleri inhibit colon carcinoma growth in mice

407 via stimulation of host antitumor immune responses. PLOS One, 12(4), 1-21.

408 Jesse, A. F. F., Tijjani, A., Adamu, L., Chung, T. E. L., Abba, Y., Mohamed, K.,

409 Saharee, A. A., Haron, A, W., Sadiq, M. A., and Mohd, A. M. L. (2014).

410 Pneumonic Pasteurellosis in goat. Iranian Journal of Veterinary Medicine,

411 8(4), 293-296.

412 Kanamarlapudi, S. L. R. K., & Muddada, S. (2017). Characterization of

413 exopolysaccharide produced by Streptococcus thermophilus CC30. BioMed

414 research international, 2017.

415 Kim, M.-J., Yang, K.-W., Yang, E.-J., Kim, S. S., Park, K. J., & An, H. J. (2015). Citrus

416 unshiu flower inhibits LPS-induced iNOS and COX-2 via MAPKs in RAW 264.7

417 macrophage cells. Oriental Journal of Chemistry, 31(4), 1915-1922.

418 Lei, Z., Zhang, F., Li-Jun, Y., Yang, G., Qing-Fang, W., & Yu, O. (2016). EPSAH, an

419 exopolysaccharide from Aphanothece halophytica GR02, improves both cellular

420 and humoral immunity as a novel polysaccharide adjuvant. Chinese journal of

421 natural medicines, 14(7), 541-548.

422 Li, P., & Wang, F. (2015). Polysaccharides: Candidates of promising vaccine adjuvants.

423 Drug Discov Ther, 9(2), 88-93. doi: 10.5582/ddt.2015.01025

Page 19 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

424 Miquel‐Clopés, A., Bentley, E., Stewart, J., & Carding, S. (2019). Mucosal vaccines and

425 technology. Clinical & Experimental Immunology, 196(2), 205-214.

426 Noor Hidayah, I. (2014). Response of serum antibody following intranasal inoculation of

427 EPS-algae adjuvanted Pasteurella multocida B:2 vaccines. (Bachelor degree),

428 Universiti Malaysia Terengganu.

429 Noraini, O., Sabri, M. Y., and Siti-Zahrah, A. (2013). Efficacy of spray

430 administration of formalin-killed Streptococcus agalactiae in hybrid red

431 tilapia. Journal of Aquatic Animal Health, 25, 142-148.

432 Olszewska, W., & Steward, M. W. (2001). Nasal delivery of epitope based vaccines.

433 Advanced drug delivery reviews, 51(1-3), 161-171.

434 Park, J. K., Kim, Z-H., Lee, C, G., Synytsya, A., Jo, H. S., Kim, S. O., Park, J. W.,

435 and Park, Y. II. (2011). Characterization and immunostimulating activity of a

436 water- soluble polysaccharide isolated from Haematococcus lacustris.

437 Biotechnology and Bioprocess Engineering, 16, 1090-1098.

438 Piermaria, J. A., Pinotti, A., Garcia, M. A., & Abraham, A. G. (2009). Films based on

439 kefiran, an exopolysaccharide obtained from kefir grain: Development and

440 characterization. Food Hydrocolloids, 23(3), 684-690.

441 Ramnani, P., Chitarrari, R., Tuohy, K., Grant, J., Hotchkiss, S., Philp, K., Campbell.

442 R., Gill, C., and Rowland, I. (2012). In vitro fermentation and prebiotics

443 potential of novel low molecular weight polysaccharide derived from agar and

444 alginate seaweeds. Anaerobe, 18, 1-6.

445 Raposo, M. F. de J., de Morais, A. M. B., and de Morais, R. M. S. C. (2015). Marine

446 polysaccharide from algae with potential biomedical applications. Marine

447 Drugs, 13, 2968-3028.

448 Razali, F. N., Ismail, A., Abidin, N. Z., & Shuib, A. S. (2014). Stimulatory effects of

449 polysaccharide fraction from Solanum nigrum on RAW 264.7 murine

450 macrophage cells. PloS one, 9(10), e108988.

451 Roier, S., Fenninger, J. C., Leitner, D. R., Rechberger, G. N., Reidl, J., and Schild, S.

452 (2013). Immunogenicity of Pasteurella multocida and Mannheimia

453 haemolytica outer membrane vesicles. International Journal of Medical

454 Microbiology, 303, 247-256.

Page 20 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

455 Sahoo, S., Chakraborti, C. K., & Mishra, S. C. (2011). Qualitative analysis of controlled

456 release ciprofloxacin/carbopol 934 mucoadhesive suspension. Journal of

457 advanced pharmaceutical technology & research, 2(3), 195.

458 Sajna, K. V., Sukumaran, R. K., Gottumukkala, L. D., Jayamurthy, H., Dhar, K. S., &

459 Pandey, A. (2013). Studies on structural and physical characteristics of a novel

460 exopolysaccharide from Pseudozyma sp. NII 08165. International Journal of

461 Biological Macromolecules, 59, 84-89.

462 Sarkander, J., Hojyo, S., & Tokoyoda, K. (2016). Vaccination to gain humoral immune

463 memory. Clinical & translational immunology, 5(12), e120.

464 Siti Aisha, M. R. (2018). Anti-inflammatory activities of compound isolated from sponge

465 associated bacteria. (Master Master thesis), Universiti Malaysia Terengganu.

466 Sompayrac, L. M. (2019). How the immune system works (2 ed.): Wiley-Blackwell.

467 Sutherland, I. W. (2001). Biofilm exopolysaccharides: a strong and sticky framework.

468 Microbiology, 147(1), 3-9.

469 Svindland, S. C., Jul-Larsen, A., Pathirana, R., Andersen, S., Madhun, A., Montomoli,

470 E., Jabbal-Gill, I., and Cox, R. J. (2011). The mucosal and systemic immune

471 responses elicited by a chitosan-adjuvanted intranasal influenza H5N1

472 vaccine. Influenza and Other Respiratory Viruses, 6(2), 90- 100.

473 Wang, B. (2011). Chemical characterization and ameliorating effect of polysaccharide

474 from Chinese jujube on intestine oxidative injury by ischemia and reperfusion.

475 International Journal of Biological Macromolecules, 48(3), 386-391.

476 Wang, S., Liu, H., Zhang, X., & Qian, F. (2015). Intranasal and oral vaccination with

477 protein-based antigens: advantages, challenges and formulation strategies.

478 Protein & cell, 6(7), 480-503.

479 Xiao, R., & Zheng, Y. (2016). Overview of microalgal extracellular polymeric substances

480 (EPS) and their applications. Biotechnology advances, 34(7), 1225-1244.

481 Xing, H., Lu, M., Xian, L., Zhang, J., Yang, T., Yang, L., & Ding, P. (2017). Molecular

482 weight determination of a newly synthesized guanidinylated disulfide-containing

483 poly (amido amine) by gel permeation chromatography. asian journal of

484 pharmaceutical sciences, 12(3), 292-298.

Page 21 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

485 Yang, J., Zhao, J., & Fang, Y. (2008). Calorimetric studies of the interaction between

486 sodium alginate and sodium dodecyl sulfate in dilute solutions at different pH

487 values. Carbohydrate research, 343(4), 719-725.

488 Yang, Y., Wei, K., Yang, S., Li, B., Zhang, Y., Zhu, F., . . . Zhu, R. (2015). Co-adjuvant

489 effects of plant polysaccharide and propolis on chickens inoculated with

490 Bordetella avium inactivated vaccine. Avian Pathology, 44(4), 248-253.

491 Zaman, M., Chandrudu, S., & Toth, I. (2013). Strategies for intranasal delivery of

492 vaccines. Drug delivery and translational research, 3(1), 100-109.

493 Zamri-Saad, M., Effendy, A., Israf, D., & Azmi, M. (1999a). Cellular and humoral

494 responses in the respiratory tract of goats following intranasal stimulation using

495 formalin-killed Pasteurella haemolytica A2. Veterinary microbiology, 65(3),

496 233-240.

497 Zamri-Saad, M., Maswati, M.A., Effendy, A.W.M. and Jasni, S. (1999b). Changes in the

498 lungs of goats with acute pneumonia following experimental challenge with

499 Pasteurella haemolytica and Pasteurella multocida. Jurnal Veterinar Malaysia,

500 11(2), 67-70.

501 Ziadi, M., Bouzaiene, T., M’Hir, S., Zaafouri, K., Mokhtar, F., Hamdi, M., & Boisset-

502 Helbert, C. (2018). Evaluation of the Efficiency of Ethanol Precipitation and

503 Ultrafiltration on the Purification and Characteristics of Exopolysaccharides

504 Produced by Three Lactic Acid Bacteria. BioMed research international, 2018.

505

Page 22 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

1

23456789

10111213141516171819

Figure number Figure titleFigure 1 The photomicrograph of a) cultured Chlorella sp. used in this

study and b) the physical appearance of EPS extracted from cultured Chlorella sp. as freeze-dried powder. Total magnification = 400x (Scale bar = 50 µm).

Figure 2 The chromatogram molecular weight of EPS extracted from Chlorella sp. using Gel Permeation Chromatography (GPC).

Figure 3 FTIR spectrum of crude EPS extracted from Chlorella sp. showing the typical structure and functional groups

Figure 4 SEM image of freeze-dried powder of EPS extracted from Chlorella vulgaris. Total magnification 2500x (Scale bar = 10 µm).

Figure 5 The graphs showed percentage relative viability of RAW 264.7 macrophage cell that was treated with various concentrations of EPS diluted in DMEM (100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.56 µg/mL). The control group was treated with complete DMEM only. Values are mean ± SEM, (n=3); One-way ANOVA was performed followed by Tukey’s multiple comparisons test for analysis.

Figure 6 Figure SEM images of exopolysaccharide (EPS) mixed with sterile PBS as excipient able to capsulate M. haemolytica A2 vaccine seed. Total magnification 6,1500x (Scale bar = 1 µm). White arrow: Fragment of Exopolysaccharide, Black arrow: Mannheimia haemolytica A2 (MhA2) vaccine seed

Figure 7 The pattern of responses of IgM, IgG and IgA in Sprague Dawley for each week of experiment. Values are mean ± SEM, (n=3) rats per group; One-way ANOVA was performed using the Tukey’s multiple comparisons test for analysis.

Page 23 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

2021

2223 Figure 1 The photomicrograph of a) cultured Chlorella sp. used in this study and b) the 24 physical appearance of EPS extracted from cultured Chlorella sp. in freeze-dried powder. 25 Total magnification = 400x (Scale bar = 50 µm).26

a) b)

50 µm

Page 24 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

2728

293031 Figure 2 The chromatogram molecular weight of EPS extracted from Chlorella sp. using 32 Gel Permeation Chromatography (GPC).3334

Page 25 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

3536 Figure 3 FTIR spectrum of crude EPS extracted from Chlorella sp. showing the typical 37 structure and functional groups 38

Page 26 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

394041424344454647484950515253545556 Figure 4 SEM image of freeze-dried powder of EPS extracted from Chlorella vulgaris. 57 Total magnification 2500x (Scale bar = 10 µm).58

Page 27 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

5960 Figure 5 The graphs showed percentage relative viability of RAW 264.7 macrophage cell 61 that was treated with various concentrations of EPS diluted in DMEM (100 µg/mL, 50 62 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.56 µg/mL). The control 63 group was treated with complete DMEM only. Values are mean ± SEM, (n=3); One-way 64 ANOVA was performed followed by Tukey’s multiple comparisons test for analysis.65

Page 28 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

666768 Figure 6 SEM images of exopolysaccharide (EPS) mixed with sterile PBS as excipient 69 able to capsulate M. haemolytica A2 vaccine seed. Total magnification 6,1500x (Scale 70 bar = 1 µm). White arrow: Fragment of Exopolysaccharide, Black arrow: Mannheimia 71 haemolytica A2 (MhA2) vaccine seed.72

Page 29 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

73747576

77 Figure 7 The development patterns of IgM, IgG and IgA responses in Sprague 78 Dawley for each week of experiment. Note that EPS-MHA2 vaccine: 79 Exopolysaccharide-M. haemolytica A2 vaccine, MHA2 vaccine: M. haemolytica A2 80 vaccine, EPS: Exopolysaccharide, PBS: Phosphate buffered saline. Values are mean ± 81 SEM, (n=3) rats per group; One-way ANOVA was performed followed by Tukey’s 82 multiple comparisons test for analysis.

Page 30 of 30

For Proof Read only


123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

Documents

For Review Only › sjstweb › Ar-Press › 2020May › 31.pdf · Sheikh, Hassan; Universiti Malaysia Terengganu, Faculty of fisheries and food Science Abdul Wahid, Mohd Effendy;