KHOA HQC CdNG NGHl
IVGHIEM CLJU TAO KHAIMG THE IgY KHAIVG l/ibria parahaema/yticus
Vo Diic Duy\ Phan Thudng T y , Phan Thi Tam', Nguyen Vd Huu PhiicS Ddo XuSn Phit' T6MTAT
Su bung phat cua hoi chiing chdt sdm (EMS) da din den nhung thiet hai nghiem frpng cho nghd nudi torn the chan frdng d Vi^t Nam va die gidi. Tac nhan chinh gay benh dupe xac dinh la vi khuan Vibrio parahaemolyticus. Cac bien phap kiem soat bang hoa chat va khang smh co the gay ra nhung tac dong Ueu cue den m6i trudng va nguoi heu diing. Vi vay, nham phat tnen ben vung nghe nuoi tOm, nghien ciiu san xuat khang the IgY phdng va tri hpi chiing EMS da duoc thuc hi^n. Nghien cmi da tidn hanh xac dinh lupng khang nguyen bat boat va loai ta chat phdi frdn cho hieu qua tao khang the IgY tdt nhat bang each danh gia eac chi heu nhu phan ling phu, nang suat de tning, luong khang the IgY tach chidt duoc tir mdi qua tning ga v^ hi6u gia ciia khang thd tach chiet dupe. Ket qua nghien ciiu cho thay ga mai gidng Novo White dupe tiem duoi da bang huyen dich khang nguyen co lupng khang nguyen bat boat 10^ CFU/mL va phdi fron bang ta chat Montanide""^' ISA 71 R VG eho hieu qua tao khang the IgY tdt nhat, voi lugng khang thd len den 105 - 118 mg IgY/tning, hieu gia khang the dat 1:4096, duy tri trong khoang 42 ngay, voi nang suat de tning dat 89%. Cac k^t qua nghien ciiu md ra tridn vong tiep tuc phat tnen san xuat cac san pham chiia khang the IgY de phong va tn hoi chung EMS fren torn the chan trang a Vif t Nam.
'Tii\ik6B.: Hoi chung cbet sdm, khang the IgY, ta chat, tom the chan trdng, Vibrio parahaemolyticus.
I.DATVANDE
Tdm the chan frang {Litopenaeus vannamei Boone, 1931) la loai tdm nudi nude Ig xuat khau chu luc cua Vidt Nam. Cung vdi su tang nhanh vd dien tich va san luong, radi trudng nudi cung ngay cang bi d nhidm, dan den tinh hinh dich benh do vi sinh vat xay ra nhidu hon, gay ra nhung anh hudng nghiem trpng ddn tinh dn dinh va nang suat nudi tdm. Can day. Vibrio parahaemolyticus, mdt loai vi khuan phan bd rdng rai trong mdi trudng bien, duoc xac dinh la tac nhan chinh gay hdi chung chdt sdm (EMS) cho tdm the chan frang (Tran va ctv, 2013), gay thiet hai kinh td vd ciing nang nd. De kidm soat loai vi khuan nay, nhieu loai hda chat va khang smh da dugc su dung fran lan; tuy nhien, tdn du cua chiing gay ra cac tac dpng nghiSm frpng ddn mdi trudng va sue khde ngudi tidu dung, tao cac chung vi khuan khang thudc (Truong Thi My Hanh va cdng su, 2016; Drais va ctv, 2016), frong khi ty 16 tdm chet van cao.
Nham phat trien bdn vung nghd nuoi tdm, hang loat nghien ciiu vd cac lieu phap thay thd khang sinh frong phdng tn benh da va dang dugc frien khai.
Trong dd, phuong phap midn dich thu ddng bang khang the dac hieu vdi m ^ benh dugc tao ra bdi ga
(IgY) dugc danh gia la cd nhidu tiem nang ling dung vi cac uu didm nhu quy trinh gay midn dich va thu khang the don gian, lugng khang the thu dugc ldn, la su thay the cd hieu qua kmh te, d6 dang va an to^n hon cho khang the tir ddng vat cd vu (Carlander va ctv, 2000).
O ddng vat thuy san, khang thd IgY dac hidu duoc chung minh cd kha nang bao ve chdng lai Edwardsiella tarda cho ca chinh Nhat (Gutierrez va ctv, 1993), Yersinia ruckeri cho ca hdi van (Lee va ctv, 2000), V. anguillarum cho ca thom (Andrade va ctv, 2013), Aeromonas hydrophila cho ca gidc gibel Gin va ctv, 2013), khang virus gay hdi chung ddm frang WSSV cho tdm he Trung Qude (Kim va ctv, 2004) va cho tdm hiim dat (Lu va ctv, 2009), khang V.
parahaemolyticus cho tdm the chan frang (Gao va ctv, 2016). Tai Viet Nam, mdt sd nghien ciiu da img dung khang the IgY frong phdng va tri bdnh d ddng vat thtiy san nhu: Khang E. ictaluri gay benh gan than mu cho ca fra (Nguydn Thi Hidn va ctv, 2013).
Do dd, nham thay the khang sinh frong phdng va tn hdi chiing EMS do V. parahaemolyticus frdn tdm tiie chan tiing, khang die IgY khang K parahaemolyticus d% dugc nghidn cuu san xuat.
Vipn Nghien ciiu Nudi frong Thiiy san ! Email; daoxu;inph;[email protected]
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 4/2020
KHOA HOC C d N G NGHfi
2 . nidi DUNG, MGUYBU VAT L f U VA PHUONB PHAP NGHBU CUU
2.1. Ndi dung nghifin cihi
- Anh hudng cua lugng khang nguyen V.
parahaemolyticus bat hoat den hieu qua tao khang thd IgY.
- Anh hudng cua 2 loai ta ch^t Freund (FCA va FIA) va Montamde™ ISA 71 R VG den hieu qua tao khang thd IgY.
2.2. Nguydn v4tlieu
- Chiing vi khu^n V. parahaemolyticus 13-028/A3 (Tran va ctv, 2013) dugc Khoa Thuy san, Trudng Dai hpc Can Tho cung cap.
- Ta chat Freund (FCA va FIA) (Thermo Fisher Scientific, My) va Montanide™ ISA 71 R VG (MI71R) (Seppic, My).
- Ga mai gidng Novo White 25 tuan tudi (can nang khoang 1,8 - 1,9 kg/con).
2.3. Phuong phap nghidn cuu
- Chuan bi khang nguyen: Vi khuan dugc nudi frong mdi trudng Manne Broth (Difco, My) d 37°C, 18 gig; sau dd, sinh khdi dupe thu va bat hoat bang formaldehyde 0,5% frong 24 gid. Sinh khdi dugc nia va huyen phii frong dem PBS (Phosphate buffered sahne, pH 7,4; Fishman va Berg, 2018) va bao quan o 4°C ddn khi sii dung.
- Phdi frpn khang nguyen vdi ta chat: Dich vi khuan bat hoat dupe trdn thu cdng vdi ta chat tao thanh huydn dich khang nguyen dung de gay midn dich cho ga.
- Gay midn dich cho ga: Ga dupe gay rmdn dich bang each tiem dudi da 1 mL huydn dich khang nguyen (9 con ga/nghiem thiic, lap lai 3 Mn), nhdm ddi chting tidm PBS. Viec gay midn dich dugc lap lai vao tuan thii 2 va 3. Cac nghi6m thiic cua timg thi nghiem dugc bd tri nhu bang 1. Hang ngay, tning ga dugc thu hoach, danh dau va bao quan d 4°C.
Bang 1. B6 tri thi nghidm gay mi&n dich cho gd Thi nghifem 1 CTNl): Anh huong cua luong khang nguyen Nghidm thuc
1 2 3 4 Doi chiing
Ta chat
MI71R
Luong khang nguyen (CFU/mL) W 10*
IQfi 10' PBS
Chi heu danh gia: N ^ g suat de tning, luong khang the m6i tning, hieu gia khang the.
Thi nghiem 2 CrN2): Anh huong cua ta chat Nghiem thiic
1 2 Ddi chiing
Ta chat MI71R
FCA (Tuan 1) FIA (Tuan 2, 3)
Luong khang nguyen Ket qua TNI PBS
Chi ti6u danh gia: Phan ling tai vi tri tiem. nang suat de tning. luong khang the m6i tning.
hieu gia khang the.
- Xac dinh nang suat de tning (%): Sd tning de dirge tiling binh mdi ngay cua timg nghiem thiic.
- Danh gia phan ling tai vi tri tiem: Kich thudc va bidu hi6n vung sung tai vi fri tidm dugc danh gia theo phuong phap cua Fodey va ctv (2008) vao cac ngay thii 7,14, 21 vdd: 0 = binh thudng; 1 = sung nhe 0 - 1 cm; 2 = sung 1 - 2 cm; 3 = sung ldn > 2 cm; 4 = sung ldn > 2 cm va phu ne.
- Tach chidt, tinh sach khang the IgY: Khang the IgY dugc tach chidt, tinh sach bang bd kit Pierce"' Chicken IgY Purification Kit (Thermo Scientific).
- Kiem fra dp tinh sach cua dich chidt IgY: Dp tinh sach cua dich chiet IgY dupe kiem fra bang phuong phap dien di SDS-PAGE (Laemmh, 1970) su dung gel cd 3% va gel tach 10%. Mau duoc frdn voi dem nap mSu 5 x theo ti le 4:1. (2% SDS, 6,25 mM Tns, 596 2-mercaptoethanol, pH 6,8). Sau khi nap mau
KHOA HOC CONG NGHE
vao cac gidng, dien di d 70V, 180 phut vdi thang chuan la BLUeye Prestained Protem Ladder (Sigma).
Gel sau dien di dugc nhupm bac theo quy trinh ciia Chevalletva ctv (2006).
- Xac dinh ndng dp khang the IgY tdng sd: Ndng do IgY frong dich chiet IgY duoc xac dinh bang bp kit IgY Chicken ELISA Kit (abeam®).
- Xac dinh hieu gia khang the IgY: Hieu gia khang the duoc xac dinh bang phan ung ELISA gian tidp (Punyokun va ctv, 2013). Cac budc thuc hien nhu sau: Khay ELISA dupe phu bing vi khuan V.
parahaemolyticus mat do 10^ CFU/mL, 25 pL/gidng va u qua dem d 37°C. Rua khay 5 lan bang PBS chua 0,05%Tween 20 (PBS-T), 200 pL/gidng. Che phu cac vi tri khdng gan khang nguyen bang 100 pL/gidng dung dich PBS-BSA 1% a 3TC frong 60 phiit. Rua khay 5 lan bang PBS-T 0,05%. Pha loang dich chiet IgY thanh cac nong dp khac nhau bang PBS theo phuong thiic pha b a n g bac 2 (1:32 - 1:8192), sau dd cho 50 pL dich chiet khang thd IgY vao timg gidng, u
kDa M 1 2 135
100 75 4S 35 25 20
d 37°C frong 60 phiit. Riia khay 5 Ian bang PBS-T 0,05%. Thdm vao mdi gidng 50 ^L dung dich cdng hgp khang flie flid khang IgY ga gan enzym peroxidase (ab6753, abeam®). Sau khi u cr 37°C frong 60 phut, rira khay va thdm 100 nL/gidng dung dich co chat OPD ndng dp 0,5 mg/mL. u d nhiet dp phdng, frong bdng tdi frong 30 phiit. Mat dp quang cua cac gidng duoc do bang may dpc ELISA Infinite F50 (Tecan, Ao) dbudc sdng 450 nm. Dich chidt IgY tir ga tiem PBS dugc su dung la ddi chiing am. Hi6u gia khang the duoc bidu thi bang dd pha loang tdi da khi OD mau/OD ddi chiing am > 2,1.
- Phuong phap xii ly sd lieu: Cac sd lieu thu thap dugc xu Iy bang phan mdm Microsoft Excel 2013 va phan mem tiidng ke SPSS 24 vdi muc y nghia P <
0,05.
3. KET QUA VA IHAO LUAN
3.1. DO tinh sach ciia dich chidt IgY
Chuoi nang (75 kDa)
Chuoi nhe
^ ^ ^ (30 kDa)
M: thang chuan protein: Lane 1, 2: IgY tu phuong phap Kit Thermo; Lane 3,4: IgY tir phuong phap PEG;
lane 5, 6: IgY tii phuong phap Na^Oj.
Hmh 1. Didn di SDS-PAGE dich chidt tiid IgY tii Idng dd tiTing ga Kdt qua di6n di SDS-PAGE cac mau dich chidt
IgY bang cac phuong phap khac nhau (Hinh 1) cho thay khang thd IgY gdm 2 phan doan la chudi nang 75 kDa va chudi nhe 30 kDa vdi dp tinh sach cao.
3.2. Anh hudng cua lugng khang nguy6n ddn hidu qua t?io khdng thd IgY
Ket qua d hmh 2 cho thay huydn dich khang nguyen cho hieu qua tao khang the IgY d ga Novo AVhite tdt, nhung dong thdi cung gay anh hudng den nang suat de tning ciia ga vi the anh hudng true tiep den san luong IgY tdng sd. Xet ve lupng khang the IgY tach chidt dugc tir mdi qua tning va hidu gia cua
khang the, khdng cd su khac biet y nghia (P > 0,05) giiia 3 nghiem tiiiic 10^ 10^ va 10' CFU/mL. vdi ngudng tdi da tuong ling la 105 - 118 mg (khoang tir ngay 21 - 77) va 1:4096 (tii ngay 35 - 84); lugng khang the IgY/tiiing va hieu gia khang tiid cua nghiem tinic 10^ CFU/mL thap hon (98 mg, 1:2048) va cd su khac biet y nghia (P < 0,05) vdi 3 nghidm thiic fren. Tuy nhien, mac dii 2 lugng khang nguy6n 10^ va 10' CFU/mL cho lugng khang tiid IgY/tiiing va hieu gia khang tiie cao nhung ddng thdi 2 lugng khang nguyen nay cung lam hit giam dang ke nang suat de tiling cua ga (P < 0,05) trong '^udt giai doan luong khang tiie va hi^u gia khang tlu dat ngudng
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 4/2020
KHOA HQC C 6 N G NGHE
tdi da (hi ngay 35 ddn 77, nang suiit de tiling trung dinh la cho hieu qua tao khang the IgY d ga Novo binh chi d^t khoang 78% va 65%) so vdi 2 lupng White tdt nhztt vdi lucmg khang the IgY/tning, hieu khang nguydn 10^ 10^ CFU/mL (nang suat de tiling gia khang the va nang suat de tning dn dinh hrong dat 89%). Chinh vi vay, huydn dich cd lugng khang ling la 105 - 118 mg, 1:4096 va 89%.
nguydn V. parahaemolyticus 10^ CFU/mL dugc nhan
T N I - N a n g s u a t d e tru-ng
—•— 10^4 —•— I O'^S —*— 1 C^e —•—10^7 —••—PBS
T h f r i g i a n ( N g i y )
TNI - Lifffng khing the IgY m5i trimg TNI - Hieu gia ciia khang th^ IgV
JbiA gbn (ngiy) n i i i gian (Ngiy)
Hinh 2. Anh huong cua lugng khdng nguyen ddn hieu qua tao khang thd feY 3.3. Anh hudng ciia td chat ddn hieu qua tao khdng thd IgY
TN2 - P h a n irng p h u ( N g i y 7)
a F r e i i n d B M I 7 1 R D P B S
TN2 - NiDg iuit de Inrag -•-Freiinil -^M171R PBS
S S ^ ? S 3 R F ; 5 ; ? 5 Tiiang d a n l i gia
T N 2 - L i n m g k h a n g t h e I g Y m a i t n i n g - • - F r e u n d -B-MITIR — P B S
TSI - I l i e u gia c u a kfaaog tbe IgV
Hmh 3. Anh hudng ciia td ch^t ddn hieu qua t^o khdng thd IgY
KHOA HOC CONO N G H £
Ket qua tii hinh 3 cho thay ca 2 loai ta chat deu it gay anh hudng den nang suat de tning (on dinh d 93% tir ngay 18), nguong tdi da cua lupng khang the tach chiet dugc bi mdi qua tning va hieu gia khdng the tuong ling la khoang 105 - 115 mg va 1:4096, su khac biet giua 2 nghiem thiic tidm huydn dich khang nguyen la khdng dang ke (P > 0,05). Tuy nhi6n, trong khi huySn dich khang nguyen sii dung ta chat Montanide™ ISA 71 R VG chi gay sung nhe cho ga vdi ty 16 thap (7,41% d ngay thii 7, va giam dan trong cac ldn kiem tra tiep theo), huydn dich khang nguyen su dung td chat Freund gay cdc v^t sung ldn cho gd trong ca 3 Ian Idem tra (ngay 7, 14, 21). K^t qua nay phii hgp vdi nhan dmh cua Fodey va ctv (2008) va Marcq va ctv (2015) cho rdng ta chat Montanide it gay kich thich phan ung viem lam anh hudng den dpng vat thi nghiem hon td chat Freund.
Vi vay, ta chat Montanide™ ISA 71 R VG dugc xde dinh Id loai td chat tot hon so vdi Freund.
4. KETLUAN
Gd giong Novo White dupe gdy mi6n dich bdng huyen dich khang nguy6n cd lugng khdng nguyen V.
parahaemolyticus bat hoat 10^ CFU/mL vd phdi trdn bang td chat Montanide™ ISA 71 R VG dupe xac dinh Id cho hieu qua tao khang the IgY tot nhat, vdi lupng khdng the len den 105 - 118 mg/tning va hieu gid khdng the dat 1:4096, duy tri trong khodng 42 ngdy, vdi nang suat de tning dat 89%.
TM UEU THAM KHAO
1. Truong Thi My Hanh, Pham Thi Yen, Huynh Thi My Le, Phan Thi Van, Nguydn Dinh Van, Nguydn Dinh Vinh, vd Truong Thi Thanh Vinh (2016). Hien trang su dung thudc va tinh khdng khdng sinh CLia Vibrio paiahaemolyticus gay benh hoai tu gan tuy cap d tdm tai Quynh Luu - Nghe An, Tap chi Khoa hpc - Cdng nghe Thiiy sdn, sd 4: 57-64.
2. Nguy6n Thi Hien, Le Hdng Phudc, Ngd TTij Bich Phuong, Nguydn Pham Hodng Huy, Vd Hdng Phuong, Chung Minh Lgi, Duong Thi Huong Giang (2013). Thir nghiem hieu qua bao ve cua thiic an bo sung khdng the khdng Edwai-dsiella ictaluri ddi vdi bdnh gan than mu tien cd tra Pangasianodon hypophthalmus. Tap chi Ngh6 cd sdng Ciiu Long, sd 2,118-126.
3. Andrade, F G., Eto, S. F , Fen-aro, A C. N. S., Manoto, D. T. G, Vieira. N. J., Cheinibim, A P., Ramos, S. P., Venancio, E. J. (2013). The production
and characterization of anti-bothropic and anti- crotalic IgY antibodies in laying hens: A long term experiment Toxicon 66:18-24.
4. Carlander, D., KoUberg, H., Wejaker, P.E, and Larsson, A (2000). Peroral immunotherapy with yolk antibodies for the prevention and treatinent of enteric infections. Immunologic Research 21:1-6.
5. Chevallet, M., Luche, S., and Rabilloud, T.
(2006). Silver staining of proteins in polyacrylamide gels. Nature Protocols 1(4): 1852-1858.
6. Drais, A A , Usup, G. and Ahmad, A (2016).
Antibiotic resistance of Vibrio parahaemolyOcus isolated from coastal seawater and sediment in Malaysia. American Institute of Physics Conference Proceedings 1784 (1), 020012 doi:
10.1063/1.4966722.
7. Fishman, J. B. and Berg, E. A (2018).
Isolation of IgY from Chicken Eggs. Cold Spnng Harbor Protocols doi: 10.1101/pdb.prot099150.
8. Fodey, T. L , Delahaut, P., Charlier, C. and Elliott, C. T. (2008). Comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs. Veterinary Immunology and Immunopathology 122: 25-34.
9. Gao, X., Zhang, X., Lin, L, Yao, D., Sun, J., Du, X., U, X., Zhang, Y (2016). Passive Immune- Protection of Litopenaeus vannamei against Vibrio harveyi and Vibrio parahaemolyticus Infections with Pmh-Vibrio Egg Yolk (IgY)-Encapsulated Feed.
Intemational Journal of Molecular Sciences 17: 723- 733.
10. Gutierrez, M. A., Miyazaki, T., Hatta, H. and Kim, M. (1993). Protective properties of egg yolk IgY containing anti-Edwardsiella tarda antibody against paracolo disease m the Japanese eel, Anguilla japomca Temminck & Schiegel. Journal of Fish Diseases 16(2): 113-122.
11. Jin, L J., U, X. Y, Zou, D. L , Li, S. Y, Song.
W. Q., Xu, Y. P. (2013). Protection of crucian carp {Carassius auratus) against septicaemia caused by Aeromonas hydrophila using specific egg yolk immunoglobulins. Aquaculture Research 44: 928- 936.
12. Kim, D. K., Jang, I. K.. Seo, H. C , Shin, S. 0., Yang, S. Y, Kim, J. W. (2004). Shnmp protected from WSSV disease by ti-eaUnent with egg yolk antibodies
44 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 4/2020
KHOA HOC CONG NGHt
(IgY) against a truncated iiision protein derived from 16. Marcq, C , Marher, D. and Beckers, Y WSSV. Aquaculture 237: 21-30. (2015). Improving adjuvant systems for polyclonal 13. Lee, S. B., Mine, Y. and Stevenson, R. M. W. egg yolk antibody OgY) production in laying hens in (2000). Effects of hen egg yolk immunoglobulin in te™^ of productivity and animal welfare. Veterinary passive protection of rainbow trout against Yersinia Immunology and Immunopathology 165 (1-2): 54-63.
ruckeri. Journal of Agricultural and Food Chemistry 17. Punyokun, K., Hongprayoon, R, Srisapoome, 48(1): 110-115. P. and Sirinarumitr, T. (2013). The production of anti-
14. Laemmli, U.K. (1970). Cleavage of structural ^'^"° Ii'in'eyi egg yolk immunoglobulin and proteins during the assembly of the head of evaluation of its stability and neutralisation efficacy, bacteriophage T4. Nature 227: 680-685. Food and Agricultural Immunology 24(3): 279-294.
15. Lu, Y. N., Liu, J. J., Jin, L J., Li, X. Y., Zhen, 18- Tran, H. L , Nunan, L , Redman, R. M., Y. H., Xue, H. Y., Un, Q. Y., Xu, Y. P. (2009). Passive Mohney, L L , Pantoja, C. R, Lightner, D. V. (2013).
immunization of crayfish (Frocambius ciarkiaii) with Detennination of the infectious nature of the agent of chicken egg yolk immunoglobulin (IgY) against "^ute hepatopancreatic necrosis syndrome affecting white spot syndrome virus (WSSV). Apphed penaeid shrimp. Diseases of Aquatic Organisms 105:
Biochemistry and Biotechnology 159: 750-758. 45-55.
PRODUCTION OFIgY ANTIBODY AGADJST Vibrio parahaemolyticus Vo Due Duy', Phan Thuong Ty', Phan Thi Tam",
Nguyen Vo Huu Phuc', Dao Xuan Phat' ' Research Institute for Aquaculture No.3 Summary
The outbreak of Early Mortality Syndrome (EMS) is now causing significant losses to the whiteleg shrimp farming industry in Vietnam and worldwide. Vibno parahaemolyticus is considered as a major pathogen. No effective treatment has currently been reported. Methods using chemicals and antibiotics can cause negative impacts on both the environment and the consumers' health. Hence, studymg to produce IgY antibody to prevent and treatment EMS is a promising approach for the sustainable development of shrimp culture. The aim of study was to find amount of inachvated pathogen and an adjuvant that could have the strongest IgY production ability. To do this, the study evaluated adverse effects, egg laying rate, amount of extracted IgY antibodies per egg and IgY titer Tbe results showed that Novo White laying hens were immunized subcutaneously with antigenic of 10^ CFU/mL inactivated V. parahaemolyticus and Montanide™ ISA 71 R VG adjuvant had the strongest IgY antibody production ability, up to 105 - 118 mg IgY/egg, the titer of IgY against V. parahaemolyticuswert 1:4096, lasted for 42 days, egg production rate were 89%. These results open tbe prospect of further developing of IgY products in order to confi-ol EMS in Litopenaeus vannamei'm Vietnam.
Keywords: Adjuvants, Early l\4ortaiity Syndrome, IgY antibody, Litopenaeus vannamei, Vibno parahaemolyticus.
Ngiifti phan bi^n: PGS.TS. T6 Long Thanh Ngdy nhan bM: 9/12/2019
Ng4y thflng qua phan bi^n: 10/01/2020 Ng4y duyet dang: 17/01/2020
