Phin II: CdNG NGHg VI SINH ^ ^ ^ 2 5 5
KHAO SAT KHA NANG TANG ClTdNG KICH THICH MIEN DICH TRONG TOM SU Penaeus monodon cua beta-glucan DOI Vdl virus GAY HOI
CHITNGDOM TRANG (WSSV)
V6 Long Thuan, Nguyen Duy Long, Hodng Quoc Khdnh Phong Vi sinh img dung
Van Thj Hanh
Phong Cong ngh? sinh hoc Dong vat, Vien Sinh hqc Nhi^t d&i
MdDAU
LTnh vyc nudi tdm dang phdt trien manh vdi ty le tdng trudng hang ndm dat khoang 16% trong hon thap ki qua. Tuy nhien viec phdt trien nghe nudi tdm bi han che bdi: i) Dich benh hoanh hanh nhung cac bien phdp ngdn chdn vd kiem soat b$nh van cdn ban che, ii) Chat lugng tdm bd m? vd tdm post-larvae khdng ddng nhat, iii) Ngudn thiic an cd chat lugng khdng phii hgp, iv) Qudn ly chat lugng nude khdng thich hgp. Trong nghien ciiu ndy, chiing tdi dieu che beta- glucan budc diu d quy md phdng thi nghiem, ddng thdi ciing tien hdnh thir nghiem boat tinh cua chl phim nay ddi vdi virus gay hdi chiing ddm trdng (WSSV) tren tdm sii Penaeus monodon.
VAT LIEU VA PHU'DNG PHAP
Tdm sii penaeus monodon thu nhan tir cac trai nudi tdm dugc xir ly vd nudi trong he thdng bl kinh, cho dn vd nudi dudng dudi thirc dn dugc dilu chl theo quy trinh hinh 1, virus gay hdi chiing ddm trdng (wssv) dugc sir dung cho muc dich lay nhilm, ldm tdc nhan gay benh. Virus dugc phdt hien tren tdm bdng SV- kit, phuong phdp PCR kem theo phuong phdp xac dinh hinh thdi huylt bdo tdm.
Ham lugng protein dugc xdc dinh bdng phuong phdp Bradford. Chl phim beta- glucan dugc tdch chilt tir nim men Saccharomyces cerevisiae theo quy trinh ben dudi (hinh 2):
256 HQi nghi KHOA HQC VA C O N G NGHg 2007 thifcan
(<Ia xay nhuySn)
beta-glucan (dgng bot)
chit k^t dinh (gelatin + tinh bot)
trOn chu va ISm 5m bSng nu'6'c im (nuac dun s6i a l nguOi nhi?t dO 40-50°C)
tao do dio c^n thilt vA ep vien
thu'c 5n chCfa beta-glucan
Hinh 1: Quy .trinh tgo thirc Sn vi?n chi>a beta-glucan
HuyIn pho nam men trong dung dich NaOH IM (1:5) (w/v) U trong d 90°C. 1 gid
i
Khuay d6u, de ngupi d nhi?t dp ph6ng
i
\
Li tam 4000 v6ng/10 phiit, thu cSn, rufa vdi niTdc 3 lan, trung h6a can n4y vdi dung dich acid acetic 2M. li tam thu cin
RiJa c3n thu diTdc bSng mpt lUdng thCTa acetone (4 acetone : intfdc (v/v)) nJfa lai 3 lan vdi nUdc
i
Thu c5n, trai th4nh Idp mong tren mpt tam plastic.
Say kho d tC, 8 gid Nghien b^ng miy nghien
i
KET QUA
, . x
\. Dieu che beta-glucan X\iSaccharomyces cerevisiae
Che pham Beta-glucan dugc dieu che tir Saccharomyces cerevisiae cd mau xam trang, cd miii, vi ddc trung va d dang bdt min. Hieu suat dieu che dugc trinh bay d bang 1.
Bdng 1: K§t qud d i l u c h i Beta-glucan tu> Saccharomyces cerevisiae
Sd l4n d i l u chd Trpng lu'p'ng r\km men ban d^u (g) Trpng lu'p'ng glucan kh6 thu du'p'c (g) Hi9u suit tach chiet (%)
Hi^u suat tach chiet trung binh (%)
1 50 3,50 7,00
3 150 10,67
7,11
2 200 14,80
7,40
4 300 21,07
7,02
5 500 35,51
7,10 7,13
2. Quan sat hinh thai te bao haemocyte
Day la cdng doan ndm yong phuong phap dim tdng huylt bao trong huylt hian^
torn, la co so khoa hgc ciing nhu lam tang do chinh xac cho phucmg phap. Qua day ciing nham muc dich xac dinh so bd hinh thai cua huylt bao (tl bao haemocyte) tdm. Cil^
huyet bao cd rit nhieu hinh dang khac nhau: hinh ydn, oval, mdng ngya, dudng kiiA yong khoang 5-12 pm. Mau sdc: cd mau xanh duong nhat khi bdt mdu vdi thudc nhuOfl"
giemsa. d yang thai sinh ly binh thudng (hinh 3), huylt bao cd mau xanh ddng lot.
^.
ph^n II: CdNG NGHE VI SINH
• ^ ^
fl!^liiil!rhj.iif!iiiiiiw{i(;j;i»(;(t«;^i^^
vi?Sffikis";*: 15;?:•-. .>.i :i!^*8: A:!
257
Hinh 3 . Tieu bdn Haemocyte nhugm giemsa du-di kinh hien vi quang hgc x100;
i- A: tieu bdn, B: trong budm d i m .
3. Ket qua nhiem nhan tao tren tom
Sau khi nhiem khoang 3 - 4 ngdy, tdm bat dau bieu hien vd chet do wssv. Bieu hien dugc ghi lai nhu sau: tdm bj nhiem ddm trdng cd bieu hien vdng ddm trdng dudng kinh 0,3 - 0,5mm d vimg vd dau ngyc, quanh mang, dn rat it, phan xa kem (bat rat de) (hmh 4)
Hinh 4. Mdu tdm c h i t do b?nh ddm trdng nhilm nhan t?o
4. Ket qua theo doi khi lay nhiem virus va cho an thirc an chura Beta-glucan Khao sat ti If song sot
^ Ti le sdng sot dat 1: Id ddi chung am ((-) wssv (-) glucan): ti le sdng dugc duy tri d niirc yen 90% yong suit thdi gian thi nghiem. Ld ddi chimg duong ((+) wssv (-) glucan): ti le sdng gidm manh, bdt diu tir ngay thir 3 (cdn 77,78%). hi ngdy thir 4 den thii 7, ti le song nay giam rit manh (tir 72,22% xudng cdn 0%). Ld thi nghiem ((+) wssv (+) glucan): ti le sdng cimg bdt diu giam vdo ngay thir 3 (cdn 81,82%) vd cimg gidm manh vdo cdc ngdy sau dd. Din ngdy thir 1, ti le sdng ciia 'd thi nghiem cdn 21,21% vd van cdn duy fri din ngdy thir 8 Id 12,12%. Mac dii, cd 2 Id (Id doi chiing duong vd Id thi nghiem) cd ti le sdng diu giam, nhung ti le sdng ciia Id thi nghiem ludn duy tri d mire cao hon Id ddi chimg duong. Dac biet la d ngay thir 7, ti le sdng d Id ddi chimg duong la 0%, trong khi Id thi nghiem van duy tri d mire 21,21% (hinh 5).
258 HQi nghj KHOA HQC VA CdNG NGHg 200 ^
I 120 > ' I , 100 I ,- ' I 80
t 60
i- 40 20
0
TI Lft S6'NG SOT d(;t I
> v ^ > » • - ». - ( - ) w s s v (.) G L U C A N
- ( + ) W S S V (.) G L U C A N - ( + ) WSSV (+) G L U C A N
I 1 3 4 5 6 7 !
r i L$ SONG S 6 T (j(/t n
100 SO 60 40 20 0
" - '•"' '-'-^"• •' h
^ ^ « > * •^i
. v ^ : ' . • .
\ ^ .;::
\ \ r::i . \X.-l
:. • N . ^ ' '
- ( - ) WSSV (.) GLUCAN - ( • ) WSSV (.) GLUCAN - (•) WSSV (+) GLUCAN
2 3 4 5 6 7 8 N(.iiv tit/ it{thivnt
^ Hinh 5: ti le sdng sdt cua tdm dg^ 1 Hinh 6 : ti le sdng sdt cua tom dgt 2
Ti le song sot dot 2: Ld ddi chiing am ((-) wssv (-) glucan): ti le sdng dugc duy yi 6 miic tren 88,89% trong sudt thdi gian thi nghiem. Ld ddi chimg duong ((+) wssv (-) glucan): sau 4 ngay nhilm, ti le sdng bat diu giam xudng cdn 96,67% vd giam manh yong 3 ngay tilp theo. Din ngdy thir 7, ti le sdng nay chi cdn 6,67%, va tdm chit hoan todn vao ngay thii 8. Ld thi nghiem ((+) wssv (+) glucan): ti le sdn^ bdt diu giam vao n^ay thii 5 (cdn 75%) vd cung giam manh vdo cac ngdy sau dd. Den ngay thii 8, ti le sdng ciia Id thi nghiem cdn 12,5%. Cac kit qua nay cung gin gidng nhu dgt thi nghiem, mac dii, ca 2 Id (Id ddi chiing duong vd Id thi nghigm) cd ti le sdng diu giam, nhung ti le song ciia Id thi nghiem ludn duy tri d muc cao hom Id doi chiing duong. Dac biet la d ngay thir 8, ti le sdng d Id ddi chimg duong la 0%, trong khi Id thi nghiem vin duy yi d mire 12,5% (hinh 6)
Tong huyet bao *•
Tdng huylt bdo dgt 1: Ld doi chimg ((-) wssv (-) glucan): tong huylt bao dugc duy' tri d miic 14,95 - 15,29 x 10^ huylt bdo/ml yong sudt thdi gian thi nghiem. Ld dii chiing am ((+) wssv (-) glucan): tdng huylt bdo giam vdo ngdy thir 3 (9,03 x 10^ huySt bao/ml), den ngay thir 6 chi cdn 1,3 x 10^ huylt bao/ml). Ld thi nghiem ((+) wssv (+):
glucan): tdng huylt bao giam cung vao ngay thii 3 (cdn 14,45 x 10^ huylt bdo/ml), giam manh d ngay thir 4 (chi cdn 2,85 x 10^ huylt bao/ml). Tuy nhien, tir ngay thir 5 ^ ngay thir 8, tdng huylt bao van duy tri duoc d mire tir 5,82 - 7,83 x 10^ huylt bao/ml (hinh 7). ^ J ^
Tdng huylt bao dgt 2: Ld ddi chimg am ((-) wssv (-) glucan): tong huylt bao du^c duy yi d miic 13,98 - 14,23 x lO' huylt bao/ml yong sudt thdi gian thi nghiem. Ld ii chung duong ((+) wssv (-) glucan): tong huylt bdo bit diu giam tu ngdy thir 2 {<M 12,67 X 10 huyet bdo/ml), giam manh d ngdy thu 4 (cdn 4,92 x 10* huylt bao/ml) v^
chl con 2,36 x 10 huyet bdo/ml d ngdy thii 6. Ld thi nghiem ((+) wssv (+) glucan)- tiii^
huyet bao ciing giam vao ngay thir 2 (11,87 x 10* huylt bao/ml), tilp tuc giam m "
den^ngay thir 4 (chi cdn 2,58 x 10* huylt bao/ml). tuy nhien, tir ngay thir 4 din ngay 7^ tong huyet bao van duy yi dugc d mire tir 4,71 - 6,89 x 10* huylt bao/ml. Nhu v, tong lugng haeinocyte yong huylt hrong tdm, qua 2 dgt thi nghiem, chi tieu ndy nhom cho an che pham beta-glucan ((+) wssv (+) glucan) mac du cd chilu hudng
Ph^n 11: CdNG NGHg VI SINH
259 nhimg vdn dn dinh va dugc duy yi d mire cao hon nhdm khdng an chl phim beta- glucan ((+) wssv (-) glucan) (hinh 8).
1)6 THI U I R N THlfiN l.L'l^NC IIAKMOCYTK THKO THrti (;iANd(;i I
u6 THI lIlfCN T H i f i N r.LfO.NG HAKVIOCYTi;
THEO n i r t l GLVN (Sell U
'^!^^'^~-'r- T:•••••'•:-!-~-Tmssi
[t!
Qf-lVVS'JViJ GLUCAfJ
• ',•> w r . s u (-,' G L U C A N a i y i VVSZV ;•; GLUCAN
Hinh 7: Dd thj biin thien haemocyte d g l 1 Hinh 8: D 6 thj biln thien haemocyte dgt 2
Ham lugng protein trong huyet tirong
' Bien thien ham lugng protein dgt 1: Ld ddi chiing am ((-) wssv (-) glucan): tdng hdm lugng protein hong huyet tuong duy tri d miic dn dinh trong sudt thdi gian thi nghiem (tir 21,03 - 26,95 mg/ml). Ld ddi chung duong ((+) wssv (-) glucan): tdng ham lugng protein trong huyet tuong bat dau giam vao ngay thii 3 (cdn 20,63 mg/ml) vd den ngay thir 6 chi cdn 7,67 mg/ml. Ld thi nghiem ((+) wssv (+) glucan): tdng hdm lugng protein hong huyet tuong ciing bat dau giam tir ngay thir 3 (cdn 15,76 mg/ml) va tiep tuc gidm d nhimg ngdy sau dd, den ngay thir 8 chi cdn 6,78 mg/ml. Tdng ham lugng protein trong huylt tuong ciia Id thi nghiem giam it hon vd ludn duy tri d miic cao hon tong ham lugng protein trong huyet tuong cua Id ddi chimg duong (hinh 9).
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Bl^NTHlfiiN HA.VILLfONC; I'ROTEIN HUY^T TlidNG THEO T H O I GIAN di/t I
BI^N THiftN H A M LL'ONG PROTEIN H L ' Y £ T T T / O N G T H E O THdJi GIAN d^fl II
Hinh 9: Biln thien hdm lu-gng protein dg^ 1 Hinh 10: Biin thien hdm lu-gng protein dgt 2
Biln thien ham lugng protein trong huylt tuong dgt 2: Ld doi chimg am ((-) wssv (-) glucan): tdng ham lugng protein trong huylt tuong ludti duy tri d mire dn dinh trong suit thdi gian thi nghiem (tir 23,04 - 24,21 mg/ml). Ld ddi chiing duong ((+) wssv (-) glucan): tdng ham lugng protein yong huylt hrong bit diu giam vdo ngdy thir 4 (cdn 19,64 mg/ml), tilp tuc giam d nhimg ngay tilp theo va din ngdy thir 6 chi cdn 9,56 mg/ml. Ld dii pghiem ((+) wssv (+) glucan): tdng ham lugng proteiii trong huylt tuong ciing bat dau pdni tir ngay thir 2 (cdn 18,18 mg/ml) va dao ddng thit thudng d nhimg ngdy tiep theo,
260 HQi nghi KHQA HOC VA C O N G NGHg 2007
nhung cd khuynh hudng cao hon Id ddi chimg duomg. Nhu vay, tdng ham lugng protein yong huylt tuong tdm: qua 2 dot thi nghiem, chi tieu nay d nhdm cho an che phim beta-glucan ((+) wssv (+) glucan) mac dii cd chieu hudng giam, cd liic dao ddng cao thip thit thudng nhung van duy yi d miic cao hon nhdm khdng an che pham beta- glucan ((+) wssv (-) glucan) (hinh 10).
5. Chuan doan va phat hien virus WSSV bdng phu-ong phap PCR va tren SV-kit.
Chuan dodn vaphdt hi^n virus WSSV bdng phucmg phdp PCR
Bang d^c lii^u wssv 364 bp
Hinh 11. K i t qua phu'ang phdp phdt hifn virus wssv tren tdm bdng PCR; g i i n g pos: mlu ddi chu'ng du'ang, gilng neg: m i u ddl chu'ng am g i l n g 1: (-) wssv (-) glucan, gidng 3: (+)
wssv (-) glucan, gilng 4: (+) wssv (+) glucan
Ket qua cho thay, Beta-glucan lam tang cudng tinh mien dich cho he thdng phdng vf d tdm. Cdn d Id ddi chiing duong, sy gia tang huyet bdo va cac protein mien dich khdng bii dap ndi sy tieu huyet (vo te bao haemocyte) do virus tien hanh ty nhan ban dya tren CO sd vat chat va nguyen lieu ciia te bdo ndy qua nhanh, khdng cd sy hd \iq cua Beta- glucan tir ban dau.
Chuan doan vd phat hien virus WSSV tren SV-kit.
i
Hinh 12: K i t qua phdt hi?n virus wssv tren torn bdng sv - kit t
Mau ddi chiing am (-) wssv (-) glucan am tinh. Mau ddi chiing duong ((+) wssv (-) glucan) vd mau thi nghiem ((+) wssv (+) glucan) diu cho kit qua duong tiiih. Dua vao' cudng do mau tren mang niyocellulose (hmh 12), chiing tdi kit luan miic do nhilm, benh cua 2 Id ddi chiing duong (+) wssv (-) glucan va mdu thi nghiem (+) wssv (+)1 glucan la nhu nhau. Dieu nay la hoan todn phu hgp vdi phuong phap cam nhiem. Cuii|i nhu phuong phap chan doan PCR ben yen, ket qua cua phuong phap nay khdng cd gid tri danh gia tmh tarig cudng he mien dich d tdm cua beta-glucan khi cam nhiem vuiis "^^
mgt lieu lugng cap tmh, ma chi cd gia tri danh gia phuong phap cam nhiem la thanh cdn De danh gia xac dang tinh tang cudng he miln dich d tdm cua beta-glucan d mgt U^
Ph^n II: CdNG NGHg VI SINH 261
lugng cip tmh, chiing tdi dya vdo cac chi tieu sinh ly mdu vd ti le sdng sdt ciia tdm hong mgt khodng thdi gian nhit dinh nhu phin kit qua va bien luan da trinh bay.
IV. JCET LUAN
Che pham beta-glucan dilu chl tir nim men banh mi saccharomyces cerevisiae cd hieu qua ddi^vdi viec tang cudng he miln dich vd duy tri thdi gian sdng tai mdt khoang thdi gian nhat dinh cua tdm six penaeus monodon a lilu gay nhiem cip tinh. Do dd, muc tieu ciia de tdi Id khao sat kha ndng tang cudng kich thich mien dich tren tdm su penaeus monodon ciia chl phim beta-glucan ddi vdi virus gay hdi chiing ddm trdng
(wssv). Muc tieu nay co ban thdnh cdng d quy md phdng thi nghiem.
Tom sir dung cdc he thdng khac^nhau chong lai nhimg tac ddng do mdi trudng bit Igi (de nhilm benh). Do cdc benh de lay nhiem vd chit lugng nude xung quanh phan anh thdng qua huylt tuong ciia tdm, cho rien cac thdng sd huylt tuong (nhu tdng lugng huyet bdo luu thdng trong he tuan hoan, tdng hdm lugng protein trong huylt tucmg tdm tai mot thdi diem nhat dinh, cac chi tieu nay dac biet cd lien quan din cdc thdng sd vl sinh ly, nidi trudng vd cdc ylu td cd thi gay sdc cho tdm) la mdt thdng sd nhay va cd y nghia nhat dinh ddi vdi viec danh gia mim benh vd cdc ylu to gay sdc tir mdi trudng.
TAI LIEU THAM KHAO
1. Van Thi Hanh (2001). Nghien ciiu sy phat triln ciia mdt sd Baculovirus trong tl bdo cdn yimg nudi cay in vitro va kha nang img dung trong sdn xuit thudc trir sau sinh hgc va kiem soat virus gay benh tren tdm, Luan an Tiln sT sinh hgc, Vien Sinh hgc Nhiet ddi, Tp. Hd Chi Minh.
2. Pham Van Tinh (1996). Ky thuat nudi tdm su, NXB Ndng Nghiep, trang 5 - 9.
3. Tri Thanh Thdo (2003). Chan doan benh ddm trang tren tdm sii (penaeus monodon) bdng ky thuat non-stop single-mbe semi-nested PCR (Kiatpathomchai, 2001), Khda luan tdt nghiep, Trudng Dai hgc Khoa hgc Ty nhien TPHCM.
4. Nguyen Thi Phugng (2005). Nghien ciiu vd phan tich mdt sd thdnh phin carbohydrate vd phuong phap pha buy vach te bao nam men, Khda luan tdt nghiep, Trudng Dai hgc Khoa hgc Ty nhien.
5. Walker GM (1998). Yeast physiology and Biotechnology, Wiley, Chichester.
6. Reimund S., (2003). A new non-degrading isolation process for 1,3-b-D-glucan of high purity from baker's yeast Saccharomyces cerevisiae, Switzerland.
7. Suphantharika M, Khuntae P, Thanardkit P, Verduyn C, (2003). Preparation of spent brewer's yeast -glucans with a potential application as an immunostimulant for black tiger shrimp, Penaeus monodon. Bioresourse Technology, p. 88, 55-60.
8. Thanardkit P, Khunrae P, Suphantharika M and Verduyn C, (2002). Glucan from spent brewer's yeast: preparation, analysis and use as a potential immunostimulant in shrimp feed, Worid Joumal of Microbiology & Biotechnology, p. 18, 527-539.
262 HQi nghi KHQA HOC VA C O N G NGHe 2007
9. Sugimoto, (1976). Process for Autolysis of Yeast, U.S Patent 3,961,080.
10. Kado (2000). Process for producing yeast exfract, U.S Patent 6,051,212.
11. Raa, J., (2000). The use of immune-stimulants in fish and shellfish feeds, University of Tromso, Norway.
12. T.W.Flegel (1998). Advances in Shrimp Biotechnology. Proceedmgs to the Special Session on Shrimp Biotechnology, 5'^ Asian Fisheries Forum, Chiengmai, Thailand.
13. Chang CF et al., (1999). Effect of dietary beta-1,3-glucan on resistance to white spot syndrome virus (WSSV) in postlarval and juvenile Penaeus monodon, FishAquat Organ; 36; 163-8.
14. Cheng FC, Su MS, Chen HY, (1999). A rapid method to quantify total haemocyte count of Penaeus monodon using ATP analysis, Fish Pathol.
15. Karin Van De Braak, (2002). Haemocytic defence in black tiger shrimp (Penaeus monodon), Wagening University, Netherland.
16. Hazel Wade, Vinh Viet Nguyen, Hue Thu Nguyen, (2002). Preliminary Overview of Shrimp Aquaculture in Vietnam.
17. Hong Phuoc Le, (2001). Haemocyte reactions against microorganisms in Black Tiger Shrimp (Penaeus monodon).
18. Vetvicka V., Sima P, (2004). -Glucan in invertebrate, ISJ 1: 60-65.
SUMMARY
Investigation of immunostimulation of beta-glucan for tiger shrimp penaeus monodon against virus WSSV
Vo Long Thuan, Nguyen Duy Long, Hoang Quoc Khanh, Van Thi Hanh Institute of Tropical Biology
Beta-glucan was found in yeast cell as a natural immunostimulants to reduce sfress and mortalities, to maintain good health of cultured organisms and to stimulate the non- . specific defence mechanism,'is becoming increasingly important in aquaculture. In this study, we investigate effect of beta-glucan to reducing susceptibility disease and^
infection of WSSV virus to tiger shrimp Penaeus monodon. A SV-kit and PCR analysis | system are proceeded to determine cell infected by WSSV virus and the blood cells are ' also investigate and counting during the freahnent with beta-glucan. The results shoWi on yeahnents and supplies by beta-glucan into experimental design tanks by feed^
increased survival percentage at 7"'-days freaynent with highest level is 21.2%. I
