PRODUCTION AND EVALUATION OF THE IMMUNE EFFICIENCY FOR INACTIVATED RIFT VALLEY FEVER VACCINE ADJUVATED WITH IMS 3013

Authors

Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo

Abstract

In this work seventeen balady sheep were divided into 6 groups, the first group (G1) was vaccinated S/C with binary inactivated RVF vaccine with 50% IMS 3013, the second group (G2) was vaccinated S/C with binary inactivated RVF vaccine containing 0.5% saponin with 50% IMS 3013, the third group (G3) was vaccinated S/C with binary inactivated RVF vaccine with 25% IMS 3013, the fourth group (G4) was vaccinated S/C with binary inactivated RVF vaccine containing 0.5% saponin with 25% IMS 3013, the fifth group (G5) was vaccinated S/C with inactivated RVF vaccine with aluminum hydroxide gel while the sixth group (G6) left as a control. The results revealed that RVF inactivated vaccine with IMS3013 either 50% or 25% give higher level of antibody and reaching its protective level earlier than RVF inactivated vaccine with aluminium gel and the best vaccine is RVF inactivated vaccine containing 0.5% saponin with 50% IMS 3013 where ED50 equal 0.0008/ml and gave a higher level of antibody all over the period of the test compared with that of other vaccinated groups when tested by lymphocyte transformation assay using MMT staining procedure and serum neutralization test.

Keywords


Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo

 

Production and evaluation of the immune efficiency for inactivated Rift Valley fever vaccine adjuvated with IMS 3013

(With 3 Tables and 2 Figures)

 

By

Lily S. Salama and Magda A. Kalad

(Received at 28/9/2005)

 

إنتاج وتقييم الکفاءة للقاح حمى الوادى المتصدع المثبط

باستخدام IMS 3013 کحافز زيتى مناعى

 

 للى صبحى سلامة ،  ماجدة أنيس قلد

 

استحدم فى هذا البحث عدد سبعة عشر من الأغنام وتم تقسيمها إلى ستة مجموعات. المجموعة الأولى تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى ومضاف إليه 50% IMS 3013 ، والمجموعة الثانية تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى ومضاف إليه 50% IMS 3013 ومضاف إليه 0.5% مادة الصابونين، والمجموعة الثالثة تم تحصينها بلقاح حمى الوادى المتصدع بالبينارى ومضاف إليه 25% IMS 3013 ، والمجموعة الرابعة تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى ومضاف إليه 25% IMS 3013 ومضاف إليه 0.5% مادة الصابونين، والمجموعة الخامسة تم تحصينها بلقاح حمى الوادى المتصدع المثبط بالبينارى مع الألمونيوم هيدروکسيد جل (أى بدون أى إضافات) بينما المجموعة السادسة ترکت کضابط للتجربة. أظهرت النتائج أن اللقاح المضاف إليه 0.5% مادة الصابونين  و50% IMS 3013 أعطى أحسن النتائج حيث کانت الEd50 = 0.0008 وأعطى أعلى مستوى مناعى طوال فترة التجربة بالمقارنة بالمجموعات الأخرى عند استخدام تجربتى تحور الخلايا الليمفاوية واختبار التعادل المصلى.

 

Summary

 

In this work seventeen balady sheep were divided into 6 groups, the first group (G1) was vaccinated S/C with binary inactivated RVF vaccine with 50% IMS 3013, the second group (G2) was vaccinated S/C with binary inactivated RVF vaccine containing 0.5% saponin with 50% IMS 3013, the third group (G3) was vaccinated S/C with binary inactivated RVF vaccine with 25% IMS 3013, the fourth group (G4) was vaccinated S/C with binary inactivated RVF vaccine containing 0.5% saponin with 25% IMS 3013, the fifth group (G5) was vaccinated S/C with inactivated RVF vaccine with aluminum hydroxide gel while the sixth group (G6) left as a control. The results revealed that RVF inactivated vaccine with IMS3013 either 50% or 25% give higher level of antibody and reaching its protective level earlier than RVF inactivated vaccine with aluminium gel and the best vaccine is RVF inactivated vaccine containing 0.5% saponin with 50% IMS 3013 where ED50 equal 0.0008/ml and gave a higher level of antibody all over the period of the test compared with that of other vaccinated groups when tested by lymphocyte transformation assay using MMT staining procedure and serum neutralization test.

 

Key words: Rift valley fever, vaccine, adjuvants, immune efficiency.

 

Introduction

 

Rift Valley fever (RVF) is an arthropod borne viral disease, affecting animals and human. It is an economically important viral disease and widely spread in different localities of Africa (Swanepoel and Coetzer, 1994) and Asia (Fagbo, 2002) where periodic epizoatic and epidemic waves occurred causing heavy losses among lambs, calves and human. The appearance of RVF disease in Egypt in 1977 (Imam et al., 1977) and its reappearance in 1993 (El-Gabary et al., 1994) increased the demand to develop a potent inactivated RVF vaccine.

Oil adjuvanted vaccines are commonly available for a wide variety of viral diseases. Oil emulsions release antigen over a longer period of time and produce a more pronounced increase in the immune response. In addition to trapping antigen, oil emulsions increase the circulation and trapping of lymphocytes in draining lymphoid tissue. Oil adjuvants may also affect the immune response by enhancing the physical presentation of antigen to macrophages (Vanselow, 1987). Beside, IMS3013 saponin (0.5%) is also added to the vaccine. It is a surface active substance that may enhances the presentation of antigen to immuncompetent cells. Being detergents they may act on the addition of hydrophobic moieties to proteins which enhances their uptake by lymph node sinus macrophages and movement into thymus dependent areas (Waksman, 1979).

New generation of oil adjuvants are designed to induce greater efficacy, improved stability and better safety, particularly with regard to the risk of residues following administration, they are termed Montanide IMS or Immunosol (Barnett et al., 1998).

This study was carried out as an attempt to improve the immunogenicity of the local produced inactivated RVF vaccine by using member of family Montanide IMS which is IMS 3013.

 

MaterialS and Methods

 

Animals:

1 - Mice (Swiss albino mice):

a - Adult mice:

21-28 day old mice were used for toxicity and potency test for both IMS 3013 and vaccines respectively.

b - Baby mice:

3-5 days old mice were used for safety of the prepared inactivated virus.

2 - Sheep:

a - Seventeen susceptible balady sheep, six months of age were used for evaluation of the immune response of the vaccines.

b - Twelve lambs of 5-10 day old were used for safety of the RVF vaccine with different concentrations of IMS 3013.

Virus:

RVF virus ZH-501 with a titre of 7.5 log10 TCID50/ml were kindly supplied by RVF Department, Veterinary Serum and Vaccine Research Institute, Abbasia, Cairo.

Adjuvant:

a- 2% gel was purchased from Honil Limited, London, United Kingdom. It was added to the binary inactivated Rift Valley fever virus (30%) to prepare the gel vaccine according to Eman (1995).

b- IMS 3013: it is an oil adjuvant obtained from Seppic, Paris, France. IMS 3013 adjuvant prior to formulation remained clear and produced a clear product on formulation. It was simple to formulate and produced extremely stable emulsion. It was prepared with Tris buffer V/V (buffer pH 7.6) then added with different concentrations to the antigen and low stear mixing at 250-300 rpm for 5 minutes was required (Barnett et al., 1998).

c- Saponin: it was obtained as a powder from KC hlight Ltd, England and prepared as 10% solution in double distilled water. It was kept overnight at 40C then filtrated through Seitz (E & S) filter, it was added with 0.5% to inactivated virus (Marcoss et al., 1998).

 

 

Toxicity test:

Adult mice were used for the toxicity of IMS 3013 adjuvant used in vaccine preparation. Three groups of mice (15 per each) one inoculated I/P (0.2 ml) and the second S/C (0.2 ml) while the third group was kept as control and all groups were observed for 10 days post inoculation according to OIE (2004).

Preparation of the new vaccine:

1- Inactivation of Rift valley fever virus:

RVF ZH-501 was inactivated by binary ethyleneimine according to Eman (1995),then different forms of vaccines were prepared, one with 25% aluminum hydroxide gel and the four others with (50% IMS 3013, 50% IMS 3013 + 0.5% Saponin, 25% IMS 3013, 25% IMS 3013 + 0.5% Saponin), respectively.

2- Addition of adjuvants:

a- Addition of IMS 3013 adjuvant:

IMS 3013 was added with different concentrations to the inactivated  virus as (50%, 25%) according to Barnett et al., (1998).

b- Addition of Saponin:

Saponin was added with 0.5% to inactivated virus according to Marcoss et al., (1998).

Evaluation of the vaccine:

Sterility, safety and potency tests were performed according to Protocol of OIE (2004).

Experimental design:

Seventeen balady sheep were divided into 6 groups:

Group 1: three sheep were vaccinated S/C with inactivated RVF with 50% IMS 3013.

Group 2: three sheep were vaccinated S/C with inactivated RVF containing 0.5% saponin with 50% IMS 3013.

Group 3: three sheep were vaccinated S/C with inactivated RVF with 25% IMS 3013.

Group 4: three sheep were vaccinated S/C with inactivated RVF containing 0.5% saponin with 25% IMS 3013.

Group 5: three sheep were vaccinated S/C with inactivated RVF with aluminum hydroxide gel (commercial one).

Group 6: two sheep were kept as control non-vaccinated.

   All animals were observed for 6 months post inoculation for detection of immunity.

 

 

Evaluation of the immune response:

1- Cell-mediated immune response:

It was measured through lymphocyte transformation assay using MTT staining procedure according to Tada et al., (1986).

2- Humoral immune response:

It was measured by serum neutralization test, which was done according to Walker (1975).

 

Results

 

Table 1: Results of sterility, safety and potency tests of the prepared vaccine

 

 

Type of vaccine

Sterility

Safety

Potency ED50/ml

Baby mice*

Lamb **

Binary inactivated RVF vaccine with 50% IMS 3013

Sterile

0/8

0/2

0.0008/ml

Binary inactivated RVF vaccine containing 0.5% saponin with 50% IMS 3013

Sterile

0/8

0/2

0.0006/ml

Binary inactivated RVF vaccine with 25% IMS 3013

Sterile

0/8

0/2

0.0005/ml

Binary inactivated RVF vaccine containing 0.5% saponin with 25% IMS 3013

Sterile

0/8

0/2

0.0006/ml

Binary inactivated RVF vaccine

Sterile

0/8

0/2

0.006/ml

 

The minimum permissible limit of ED50/ml is 0.02ml

* Safety test in baby mice = no signs of illness or death.

** Safety test in lambs = no thermal or clinical reaction or manifestation.

 

Table 2: Results of cell mediated immune response as tested by the lymphocyte blastogenesis and expressed by MTT assay for sheep vaccinated with different formula of RVF prepared vaccines.

 

 

Table 3: Results of neutralizing antibody index (NI) of sheep sera vaccinated with different formula of RVF prepared vaccines.



 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion

 

The progress in RVF vaccine production is directed towards the selection of the proper adjuvant that can elaborate a high and long lasting immunity. So adjuvants are considered one of the important factors in vaccine formulation.

Therefore usage of oil emulsion has taken in consideration for production of different vaccines due to strong benefit of newly oil adjuvants. A new generation of oil adjuvants termed Montanide IMS or Immunosols, have been commercially developed which form “microemulsious”.

Their composition includes new immunostimulants, listed as GRAS substances and are reported to elicit both humoral and cell-mediated immune responses. They are of a water dispersable composition and are therefore extremely fluid, physically stable for at least six months at 4oC following formulation and give no local reaction (Barnett et al., 1998).

Toxicity test on mice revealed that up to 50% of IMS 3013 were non toxic and 0.5% saponin also non toxic as it is the best percentage which can be added to the inactivated virus suspension (RVF antigen) as mentioned by Marcoss et al., (1998).

The different formula of the prepared vaccine were sterile and safe when inoculated in baby mice and lambs which showed no elevation in body temperature in lambs and no signs of illness or deaths were observed in mice and lambs (Table 1).

The most potent vaccine is that containing IMS 3013 50% with 0.5% saponin as its ED50 was 0.0008/ml as shown in Table (1).

The results of cell-mediated immune response tested by lymphocyte blastogensis assay are illustrated in Table (2) and Fig. (1).

It reveals that the T cell response occurs from the 1st day post vaccination and reaching its peak at the 10th day post vaccination in the five groups but with higher degree in groups (2 & 4) till the end of the experiment.

These results showed an enhancement of cellular immune response of sheep vaccinated with RVF vaccine with IMS 3013 as an adjuvant together with 0.5% saponin than inactivated vaccine without oil. These findings agree with Lily (1991), Eman (1995) and Marcoss et al., (2005).

The results of serum neutralization test in Table (3), Fig. (2) showed that the sera of sheep vaccinated with RVF vaccine + 50% IMS 3013 (group 2) gave the highest level of antibody response, the antibody reached the protective level at the 2nd week post vaccination (NI = 1.6) as Pini et al., (1973) suggested that the protective level was log 1.5. These results agree with that obtained by Gehan (1990) who found that sheep vaccinated with oil emulsion inactivated RVF vaccine had a high level of antibody. This also agree with Doel et al., (1994) and Salt et al., (1995) & (1997). They found protection as early as 4 days post vaccination using ISA 206 (one of the Montanide) as an adjuvant in FMD vaccine production.

Animals of group 2 and group 4 which vaccinated with RVF inactivated vaccine and containing 50% and 25% IMS 3013 respectively together with 0.5% saponin in both groups showed an antibody level which do not great differ from that of group (1 & 3) which do not contain saponin. These results agree with that obtained by Marcoss et al., (1998).On the contrary the two groups (2 & 4) showed higher cellular immune response than all other groups. So this gives an indication that saponin plays an important role through enhancement of cellular immune response.

The obtained results may be attributed to the mode of action of each adjuvant as shown by Edelman (1980) who studied the mode of action of aluminum gel as a compound which produced local granulomas that contained antibody producing plasma cells, while Herbert (1968) revealed that the action of oil emulsion as a mineral delaying absorption of antigen and stimulating mononuclear cells to produce antibodies at local sites and also increases the circulation and trapping of lymphocytes in draining lymphoid tissue. So the oil emulsion stimulates humoral immunity and cellular immunity.

From the above study, it could be concluded that RVF vaccine with 50% IMS 3013 and 0.5% saponin is the best type as its ED50 was 0.0008/ml and it gave the highest level of immunity either cellular or humoral. This agree with Ali et al., (2004) who found that IMS 3013 gave higher fifty percent protective dose of guinea pigs (GPPD50) than aluminum hydroxide gel vaccine.

It may be concluded from the present work that the application of RVF IMS 3013 vaccine will enable a reduction of the frequency of animal vaccination. IMS 3013 vaccine may be used for emergency vaccination in RVF outbreaks.

 

 

 

ReferenceS

 

Ali, S.M. and Roshdy, O.H. (2004): Comparative evaluation of the immune efficiency for FMD vaccines prepared with IMS 3013 and aluminum hydroxide gel in guinea pigs. Egyptian Vet. Med. Assoc., 64(2): 237-244.

Barnett, P.V.; Pullen, L.; Warder, P. and Slatham, R. (1998): Preliminary studies on emergency Foot and Mouth disease vaccines formulated with Motanide IMS (Immunosol), a new concept in oil adjuvancy. European Commission for the control of FMD Aldershot, United Kingdom, 14-18.

Doel, T.R.; Williams, L. and Barnett, P.V. (1994): Emergency vaccination against Foot and Mouth disease: Rate of development of immunity and its implications for the carrier state. Vaccine, Vol. 12, 7, 592-600.

Edelman, R., (1980): Vaccine adjuvants. Rev. of Inf. Dis., 2: 370-383.

El-Gabery, G.H.; Nawal, M.A.; Hadia, A.; Fathia, M.M. and Ayoub, N. (1994): Unclassical picture of RVF in man and animals in Aswan governorate in May 1983. Vet. Med. J., Giza, 42(1): 133-139.

El-Nimr, M.M. (1980): Studies on the inactivated vaccine against RVF. Ph.D. Thesis, Microbiology, Fac. Vet. Med., Assuit University, Egypt.

Eman, M.S. (1995): Studies on Rift Valley Fever vaccine inactivated with binary. Ph.D. Vet. Sc., Fac. Vet. Med., Cairo University, Egypt.

Fagbo, S.F. (2002): The involving transmission pattern of Rift valley fever in the Arabian Peninsula.

Gehan, K.M. (1990): Studies of Rift Valley Fever among animals in Egypt. Ph.D. Thesis, Infectious Diseases, Fac. Vet. Med., Zagazig University, Egypt.

Herbert, W.J. (1968): The mode of action of mineral oil emulsion adjuvant on antibody production in mice. Immunology, 14: 301-318.

Imam, Z.E.I. and Darwish, M.A.A. with technical association of El-Karamany, R. and Omar, F. (1977): A preliminary report on an epidemic of Rift Valley Fever in Egypt. J. Egyptian P.H. Ass., L III (6): 417-418.

Lily, S. Salama (1991): Studies on the immune response  of Rift Valley Fever vaccine. M.V. Sc. Thesis, Microbiology, Fac. Vet. Med., Cairo University, Egypt.

Marcoss, T.N.; Lily, s. Salama and Elian, K. Aly (1998): Studies of different adjuvants on the immune response of sheep to Rift Valley Fever inactivated vaccine. Vet. Med. J., Giza, Vol. 46, No. 4B, 719-727.

Marcoss, T.N.; Salib, O.P. and Daoud, A.M. (2005): Use of mycobacterium phlei as an immunopotentiating factor with inactivated RVF vaccine. Egypt. Vet. Med. Assoc.. 65(3): 29-37.

OIE (2004): Rift Valley Fever.

Pini, A.; Lund, L.J. and Davies, S.J. (1973): Flourescent and neutralizing antibody response to infection by Rift Valley Fever virus. J.S. Afri Z. Med. Ass., 44(11): 161-165.         

Salt, J.S.; Williams, L.; Statham, R. and Barnett, P.V. (1995): Further studies on the rate of development of protection in cattle given emergency vaccination against FMD. Report, Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot and Mouth disease and the Foot and Mouth disease subgroup of the Scientific Veterinary Committee of the Commission of the European Community, Moeldling, Appendix 17, 90-97.

Salt, J.S.; Dani, P.; Williams, L. and Barrnett, P. (1997): Efficacy studies in pigs with two novel oil adjuvanted emergency FMD vaccines. Report, Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot and Mouth disease and the Foot and Mouth disease subgroup of the Scientific Veterinary Committee of the Commission of the European Community, Israel, Appendix 23, 162-167.

Swanepoel, R. and Coetzer, J.A.W. (1994): Rift valley fever, infectious diseases of livestock with special reference to South Africa, edited by J.A.W. Coetzer; G.R. Thomson and R.C. Tustin. Cope Town: Oxford University Press, 1: 688-717.

Tada, Hiroko, Osama Shiho (1986): An improved calorimetric assay for interleukin 2. J. Immunological Methods, 93: 157-165.

Vanselow, B.A. (1987): The application of adjuvants to veterinary medicine. Vet. Bult., 57: 881-896.

Waksman, B.H. (1979): Adjuvants and immune regulation by lymphoid cells. Springer Semin., Immunopathol., 2: 5-33.

Walker, S.J. (1975): Rift Valley Fever: a review committee on foreign animal diseases, United State of Animal Health Association USA, Army Med., Bes. Inst. Inf. Dis., Fredrick, Maryland.


 

Groups of animals

Types of different adjuvants

No. of animal

Before vaccination

Days post vaccination

1st

3rd

5th

7th

10th

15th

21st

28th

G1

IMS 3013 50%

3

0.116

0.156

0.192

0.259

0.441

0.459

0.411

0.392

0.370

0.110

0.129

0.181

0.241

0.410

0.433

0.401

0.381

0.361

0.109

0.119

0.176

0.212

0.401

0.410

0.388

0.360

0.311

Mean

 

0.111

0.134

0.188

0.237

0.417

0.434

0.400

0.377

0.347

G2

IMS 3013 50% + 0.5% saponin

3

0.108

0.162

0.301

0.476

0.500

0.513

0.475

0.429

0.384

0.112

0.171

0.351

0.482

0.541

0.561

0.497

0.467

0.411

0.110

0.138

0.306

0.466

0.513

0.520

0.483

0.431

0.397

Mean

 

0.110

0.157

0.319

0.474

0.518

0.531

0.485

0.442

0.397

G3

IMS 3013 25%

3

0.114

0.115

0.157

0.201

0.265

0.301

0.287

0.248

0.220

0.101

0.109

0.110

0.189

0.248

0.290

0.278

0.210

0.201

0.107

0.110

0.127

0.192

0.219

0.285

0.259

0.238

0.192

Mean

 

0.107

0.125

0.162

0.203

0.297

0.291

0.274

0.232

0.204

G4

IMS 3013 25% + 0.5% saponin

3

0.110

0.126

0.138

0.201

0.270

0.361

0.321

0.301

0.293

0.115

0.131

0.171

0.219

0.321

0.370

0.346

0.298

0.277

0.109

0.116

0.161

0.191

0.302

0.304

0.311

0.289

0.250

Mean

 

0.111

0.129

0.184

0.213

0.319

0.345

0.326

0.296

0.273

G5

Aluminum gel

3

0.118

0.131

0.199

0.221

0.297

0.301

0.233

0.225

0.221

0.110

0.126

0.187

0.213

0.278

0.285

0.222

0.210

0.201

0.114

0.119

0.182

0.207

0.259

0.293

0.216

0.197

0.181

Mean

 

0.114

0.119

0.152

0.194

0.244

0.262

0.223

0.210

0.201

G6

Control

2

0.108

0.116

0.111

0.116

0.109

0.112

0.121

0.119

0.119

0.119

0.128

0.120

0.121

0.118

0.123

0.124

0.122

0.126

Mean

 

0.113

0.122

0.115

0.118

0.113

0.117

0.122

0.120

0.122

 

 

 

 

 

 

 

 

 

Groups of animals

Types of different adjuvants

No. of animal

Before vaccination

Weeks post vaccination

1st

2nd

3rd

4th

6th

8th

12th

16th

20th

24th

G1

IMS 3013 50%

3

0.4

1.0

1.7

2.0

2.4

2.7

3.0

3.4

3.0

2.7

2.4

0.3

1.0

1.4

1.7

2.0

2.4

3.0

3.0

2.7

2.4

2.4

0.3

1.4

1.7

2.0

2.0

2.7

3.4

3.4

3.0

2.4

2.0

Mean

0.3

1.1

1.6

1.9

2.1

2.6

3.1

3.2

2.9

2.5

2.2

G2

IMS 3013 50% + 0.5% saponin

3

0.7

1.4

1.7

2.0

2.4

2.7

3.0

3.4

2.7

2.4

2.0

0.4

1.4

1.7

1.7

2.4

3.0

3.4

3.4

3.0

2.7

2.4

0.3

1.0

1.4

1.7

2.0

2.4

3.0

3.7

2.7

2.4

2.0

Mean

0.4

1.2

1.6

1.8

2.2

2.7

3.1

3.5

2.7

2.5

2.1

G3

IMS 3013 25%

3

0.3

1.0

1.4

1.7

2.0

2.4

2.7

2.4

2.4

2.0

1.7

0.4

1.0

1.4

1.4

2.0

2.0

2.7

2.7

2.4

2.4

2.0

0.3

0.7

1.0

1.7

1.7

2.4

3.0

2.7

2.0

1.7

1.7

Mean

0.3

0.9

1.2

1.6

1.9

2.2

2.8

2.6

2.2

2.0

1.8

G4

IMS 3013 25% + 0.5% saponin

3

0.7

1.0

1.4

1.7

2.0

2.7

3.0

2.7

2.7

2.4

2.0

0.7

0.7

1.0

1.4

1.7

2.0

2.7

2.7

2.4

2.0

1.7

0.4

1.4

1.4

1.4

1.7

2.4

3.0

2.4

2.0

2.0

2.0

Mean

0.6

1.0

1.2

1.6

1.8

2.3

2.9

2.6

2.3

2.1

1.9

G5

Aluminum gel

3

0.3

0.7

1.0

1.0

1.4

2.0

2.4

2.0

2.0

1.7

1.4

0.4

1.0

1.4

1.7

1.7

2.0

2.7

2.7

2.4

1.7

1.7

0.7

0.7

1.0

1.4

1.7

2.4

2.7

2.7

2.0

2.0

1.7

Mean

0.4

0.8

1.1

1.3

1.6

2.1

2.5

2.3

2.1

1.8

1.6

G6

Control

2

0.3

0.2

0.4

0.3

0.3

0.4

0.2

0.2

0.4

0.3

0.2

0.3

0.3

0.3

0.3

0.4

0.4

0.3

0.2

0.3

0.3

0.3

Mean

0.3

0.25

0.35

0.3

0.3

0.4

0.25

0.2

0.3

0.3

0.25

 

 

 

ReferenceS

 
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