D,;.IHQC Qu6c GIA - TP. HCM TRUONGD,;.IHQC KHOAHQC TVNHIEN L YTHJ THANH LOAN " , ",," A1 NGHIEN CUU MOT SO VI KHUAN VA VIRUS GAY B~NH TREN TOM SU (Penaeus monodon) " A1 NUOITHU<1NGPHAM {j DONG BANG SONGcifu LONG CHUYEN NGANH : VI SINH MA SO: 1.05.12 TOM TAT LU!N AN TIEN si SINH HOC TP. HO CHI MINH - 2003 J>-L tONG TRiNHaliOC HoANTHANHT41 VleN NGHIENcl1u NUOITRaNGTHUYSANII Ngllirihll(fngdin khoahoc: 1. PGS.TS.PH4MTHANHHO 2~ TS.NGUYENVANHAO Phin bien1: .PGS.TS.BiJiLai , Phin bien 2: PGS.TS.Tran LinhThuoc Phin bien3: .TS.TrinhTruongGiang " ,., , Lu~nan se duQcbaav~ truDcHOidongcha'mlu~nanca'pnhanuDc hopt~i TruOngE>~ihoc KhaahocTI,lnhien , , D(,lihoc Quac gia - Tp.Ho ChI Minh vaohoi,09.giCJ00ngay30 thang 01 nam2004 Coth~tim hi~ulu~nant~ithuvi~nKhoahocTOnghOpva thuvi~nTruOngE>~ihocKhoahocTI,lnhien- TP.HoChiMinh Lu4n an "Nghien cztu m(}t so'vi khudn va virus gay b~nh tren tom su (Penaeus monodon) nuoi thlldng phdm d'D8ng Bang Song Cau Long" Chu'dng 1: MOE>AU 1.1. Tinhc6p thiet cua luin an Trong nhung th4p Bien g~n day, nu6i tr6ng tMy san c1khu vf!cCMu An6i chung va Vi~tNam n6i rieng dang tren da phat tri~n, nha'tla ngM nu6i t6m bi~n c1nhung vung veil bi~n, cii'a s6ng. Tuy nhien, mQt trC1ng~i ldn anh hu'C1ngde'n Dang sua'tva san 1u'<;1ngla va'nd~ djch b~nh bUngn6 trong qua trlnh nu6i cling vdi sf!suy thoai m6i tru'<1ng. Vi the', va'n d~ ca'p bach hi~n nay la pMi xac djnh nguyen nMn gay che't t6m nu6i, cac ye'u t6 m6i tru'<1ng,mua vt,lxua'thi~n b~nh d~ giam nhung thi~t h~;ddo ~nh t6m gay ra h~u c6 th~ mC1rQngva phat trieD ngh~ nu6i Wmbiln mQtcach Mn vung (j Vi~t Nam, ct,lth~ la cac dnh D6ng B?ingS6ng Cii'uLong (DBSCL). 1.2. Ml,lcti~u cua luin an D~ tai du'<;1cthf!Chi~n nhiim xac djnh vi sinh v4t (vi khua'n va virus) gay b~nh; hta tu6i nhi~m va th<1idilm xua't hi~n mQts6 b~nh thu'<1ng g~p tren t6m su nu6i c1cac m6 hinh nu6i cM ytu: quang canh (QC), ban tham canh (BTC), "Tom - Lua" va tham canh (TC) - nu6i c6ng nghi~p (CN) d DBSCL d~ lam cd Sc1khoa hQc cho vi~c d~ xua't cac giai phap phOngtq thfch h<;1p. 1.3. NQidung cua luin an - Nghien cU'u~m ~nh vi khua'n, cM ye'u nh6m Vibrio tren t6m su nu6i tren m6 hinh BTC va "Tom -Lua". - Nghien cU'u~m b~nh virus (MBV va WSSV) tren t6m su va cac loai thuQchQt6m he tren m6 hinh BTC, "T6m - Lua" va QC ("Tom - r ' "-""-'" " " Rang" va "Rang-Tom"). II:)H. V.i-LTt! NHift'iI , ,., ! . f U U' \. 1 p'r' r,\ ' ! ! . n \lJ '; ~1::'1\4 ! ! """":-"'\ ! . l j 2 - Nghien cti'u va M th6ng cac b~nh thu<'1ngg~p tren tom su nuoi thudng phGm(Penaeus monodon) d mo hinh nuoi TC. 1.4. Netmd!cua lu,n an - Lu~n an la mQttrong nhii'ngoong trinh d~u tieD d Vi~t Nam nghien cti'u c6 M th6ng cac vi sinh v~t gay b~nh quail trQng:nh6m Vibrio, MBV, WSSV tren tom su nuoi d cac roOhinh khac nhau (QC, BTC va TC) t~i cac dnh BBSCL. - Lu4n an ding la mQt trong nhung cong trinh dAu tieD nghien cau va tdng hqp cac b~nh thu<'1ngg~p tren tOmsu d cac giai do~n phat tri€n khac nhau tren mo hinh nuMTC. Ynghiii khoa h{Jc Hinh thanh cd sd khoa hQc cho vi~c xac dinh nguyen nhan gay che't tom nuoi d BBSCL va M xua't cac giiii phap phOng tranh. Ynghia tht/c ti5n -Giup Dongdb c6 th€ phong ngita b~nh tren tom trong chu ky nuoi thong qua vi~c qUail 19 sac khOe tom nuoi cung vdi cac giiHphap do lu4n an d~ xua't. - Ke't qua cua lu~n an giup cd quail qm\n 19Nha nudc c6 duqc cd si'1 khoa hQCva cac thong tin c~n thie't v~ nguyen nhan gay che't tom nuoi d BBSCL, nh~m ph\lc V\lchie'n luqc phOngtri b~nh tren tom nuoi thich hqp. Chuang 2: TONGQUANTAl LieU G6m 6 d~ m\lc: D~ IIlQCd~u tieD Higidi thi~u sd net v~ tinh hinh nuoi va dich ~nh tom teen the' gidi. San d6 khao sat v~ tinh hinh nuoi tom su va nhung thi~t h~i do dich ~nh tom gay fa d Vi~t Nam. D~ m\lc 2: thai quat v~ cae roOhinh nuoi tom phd bien d B6ng B~ng Song Cii'uLong (DBSCL): BTC, "Tom-Lila" , QC ("Tom - 3 Rung", "Rung - Tom") va TC(nuMcongnghi~p). E>~m\lc 3 va 4: gidi thi~u cac ~nh thu'<'1ngg~p tren tom so.nuoi va cac vi sinh v~t g~y ~nh chinh tren tom so. nhu' b~nh do virus (MBV, WSSV, YHCV); b~nh nhi~m khu~n (nh6m Vibrio). E>~m\lc 5: gidi thi~u mQtvai phu'dng phap nghien cU'uvi sinh v~t g~y b~nh tren tom so.va cac loai thuQc h<?tom he nhu': phu'dng phap cd di~n (nuoi ef{y va phan l~p tac nhan g~y b~nh), phu'dng phap huye't thanh h<?c,phu'dngphap sinh h<?cphan tii',phu'dngphap mo h<?c. E>~m\le 6: sd net v~ tinh hlnh nghien eU'u~nh vi sinh v~t (virus va vi khu~n) tren tom so.va cac loai tom he tren the' gidi va aVi~t Nam. Chlidng 3: V4T lieU vA PHUONG PHAp NGHIEN CUU 3.1. Nghi6n cuu mSm btnh vi khuin tr6n tom nuoi is eaSCl 3.1.1. Thai gian, dla di~m, IliQng miu phan trch 3.1.2. Phlidng philp - Phan l~p tae nhan g~y ~nh tir cae milu tOmbhng phu'dngphap nuoi c{{ytruy~n th6ng va dinh daub vi khu~n theo Bergey, 1994. - G~y nhi~m nhan ~o d~ xac dinh kba mlng g~y b~nh cua cae loai vi khu~n phan l~p tir cae milu tom bhng phu'dngphap ngam va tiem. - T~o khang huye't thanh cua V. harveyi tren ca tai tu'<;1ngva thii'1i'ng d\lng trong ch~n doan nhanh vi khu~n g~y ~nh tren tom (theo phu'dng phap eua cae tae ghl J.A. Plum va P.R. Bowser (1982); Grimes va etv. (1987); Yii va etv (1997). Thiti wan Dia diim S61u'nl! m u thu Mcj hlob 02/1994 - 03/95 Cae tinh f>BSCL 256 mu Ban tham eanh 02/97 - 05/97 Cn f>u"de- 102 mu "Tom-Lila" Long An 4 3.1.3. XU'IVs61i~u Ap d\mgcacph~nm~mtrongchu'dngtrinhSPSSde xii'19s6lic$u. Dung phep rinhth6ng ke mo ta de rinhcac gia tIi: gia tIi trungbiBb (mean),dQlc$chchu~n(SD),M s6 tu'dngquaDPearson va phu'dngsai ANOVAde timcacm6itu'dngquaD. 3.2. Nghi6n ct1u mim b~nh virus: virus g6y b~nh col (MBV)va virus g6y hoi chang d6m trAng (WSSV)tr6n t6m su va cae loai is hQt6m he 3.2.1. ThCligian, dla (liim, htC1ngmiu ph6n tfch Ghi eM: (*): phdn tfchMBVva WSSV bdng phllang phdp mo hqc truy€n tMng. (**): phlin rich MBV bdng phllang phdp mo hqc truyin tMng; WSSV bdng phllang phap nW hqe va PCR. 3.2.2. Phu'dng phap - Phltdngphdp M6 hqc: m§u tom du'<;1cthu ph~n gall tl}.yva mang (m§u tOmgi6ng thu nguyen con) c6 djnh trong dung djeh Davidson. Xii'19, duc trong parafin, cAthit vdi dQday 5 - 6 J1m;nhuQmvdi Hematoxylin va Eosin. 8ge ke't qua theo Lightner (1996a). - Phltdng phdp PCR: mall thu pMn mang (mau tom gi6ng thu nguyen con) c6 djnh trong cdn 95%, du'<;1cnghi~n vdi dung djeh SDS (Sodium Dodecyl Sulphate) 0,0025% va NaOH 0,005N de ly trieh DNA. San d6 tie'nhanh pban 6'ngPCR theo quy trinh cda Kim (1998). - Phltdng phap phdn richcae chi tieu chtlt Iltqng nltoc: pH, 00, dQtrang, dQm~n du<;1e de djnh theo cae phu'dng phap thong thu'<7ngcua phong thi nghic$m thuy h6a. ThO'iIdan Dia diim S6miuthu Me)hlnh to'02- Cn Dude - 108 mfiu "TOm-LUa" 05/1997 Long An (*) to'03- Duyen Hili - 91 mfiu BTC 07/1998 Tri'iVinh (*) to'06/97 - Ci'iMau (**) 72 mfiu: TOmgitfng "Tom-Rimgn, 05/98 216 mfiu: TOm trwng thanh "Rimg-Tom" 5 3.2.3. XU'IVsoU,u Ap d~ng cae ph~n m~m trong chu'dng trlnh SPSS d~ xii' ly s6lic$u. 3.3. H, thong mQtso b,nh thuC1ngg4p tr~n t6m su Csm6 hlnh nu6iTC 3.3.1. ThC1igian, dla diim, so ao nghh!n euu Thitigian I Dia di~m I S6 ao n2hi~n eU'u 2 ao D~e di~m 1999-2000 1 Long Hlidng (Bi! Ria - VfingTau 1999-2000 IC6nC6ng (TienGian. B<;1eLi~u 1999-2000 VinhH~u 03-07/2001 Trai Thlien hiem 3.3.2. Phuong phap nghi6n euu - Quan sat da'u hic$ubc$nhtich va bi~u hic$nlam sang cae bc$nh: + Lien quaD de'n ye'u t6 moi tru'C1ng:bc$nhmang barn, b~nh nhiem khuei'n,bc$nhden mang, hQich6'ngtom n6i d~u. + Cae bc$nhdo virus: bc$nhMBV, bc$nhd6m trilng (WSD). - Quan trilc cae ye'u t6 moi tru'C1ng: + Nhic$tdQ,pH, oxy bOa tan (sang va chi~u). + Do NH3-N,COD, de)~n: 2 tu~nll~n. - Thu m§u tOm: 10 ngayllftn, kiSm tra mftm b<$nh WSSV va MBV. Chuang 4: KET QUA -THAo Lu4N 4.1. Nghi6n euu mim b,nh vi khu§n tr6n t6m nu6i CseBSCL 4.1.1. Tin so xuat hi,n ella cae loi:tiVibrio e6 kha n4ng gay b,nh tr6n t6m nu6i Csm6 hlnh BTC 4 ao 22 ao 2 ao Vung da't cao trieu, yen bi€n Nam 86m!.Hau 6 Klt qua ghi nh~n c6 Sf!.sai khae y nghla (p <0,05) v~ ~t th6ng k~ giira cln so' xu!t hi~n cia cae loai vi khua'n g;iy ~nh: V. parahaemolyticus, V.alginolyticus, V.harveyi tr~n cae mill t6m e6 ddu hi~u b~nh d~u caDhdn cae mill kh6ng c6 ddu hi~u ~nh. Bang 4.1.1 T~ns6 xua'thi~ncda cae loai vikhuln tren eae mAll tom nuoi- MohlnhBTC41icaetInhDBSCL n: s6 milu kilm Ira 4.1.2. Tin 66 cua cae loBIVibrioco kha Ding gay btnh t~n t6m 6U nu61 iJ m6 hinh "T6m -Lua" Bang 4.1.2 T~n s6 cda eae 10Mvi khu~n Wn cac m§u tom nudi - Md hinh "T8m - Laa" ta i C~n Dltdc -LongAn Ghi eM: n: s6 mdu kitm Ira Tan s6 xucft hien (%) Loai vi khudn TrJmkMng co dt1uhifU TrJmco dt1uhiu nh benh (n =15) (n =24]) Vibrio so. 100 100 V. oaraheamolvticus 0 2282 V. alfdnolvticus 0 224 V. anf!uillarum 0 39,8 V. cholerae non 01 200 9.9- V.harvevi 0 4.1 V. oelafdus 0 236 V.metschnikovii 15 0 V.fluivalis I 866 58 Pseudomonas so. 600 481 Enterococcus 666 1,2 Loai vi khulin TAnslf xulft hi2n (%) Mdu nh (n =41 ) Mdu dink IcY(n =41) Vibrio so. 100 100 V. alrdnolvticus 26.83 17.07 V. anf!uillarum 26.83 2.43 V. cholerae 26.83 4.87 V. harvevi 2683 12.19 V. vulnitfcus 0 31.70 V.camobelli 34.15 29.26 V. mimicus 0 78.04 V. fluivalis I 55,65 14,63 7 Ke't qua khao sat cae m~u t6m tho t~i C~n Dude -Long An, ghi nh~n cae loai vi khu~n g~y b~nh hi~n di~n trong cae m~u tho d<;Jtdieh b~nh eho ke't qua eao hdn cae m~u tho dinh ky. xU' 19th6ng ke eho tha'y e6 sf/.sai khae 9 nghia (p < 0,05) d6i vdi cae loai V. harveyi, V. cholerae, V. anguillarum gitYacae m~u tho dinh ky va tho Ilk dieh ~nh. 4.1.3. Xac dlnh kha n4ng gay b,nh cua cae lOBiVibrio tren t6m - Ke'tqua gay nhi~m ghi nMn khciDang gay ~nh eua cae loai Vibrio g~y nhi~m tren t6m, dii ehti'ng minh c6 m6i tudng quaD gitYacae loai Vibrio g~y ~nh vdi cae m~u t6m c6 bilu hi~n b~nh deh dii tho du'<;Je tren cae ao nu6i nhu':do than, phdng v~y r~u, ehan du6i, ho~i tU'cae pMn ph~. -Ke't qua gay nhi~m ghi nMn ty l~ t6m ehe't b?1ngphu'dngphap tiem eao hdn phu'dng phap ngam. Di~u nay eho phep ghi nh~n t6m nhi~m khu~n huye't (nhi~m khu~n toau th~n) thi g~y tae h~i hdn t6m bi nhi~m khu~n e~e bQ. Bang 4.1.3 TY11$tOmche'ttrungblnhsankhig~ynhi6m168gio (7ngay) Loai vi khu4n Phddng ph8p gay Ty I tm nh va chtt nhim saD 168 fliit (7 nflAy) V. harveyi nf1;m 70,00 :t 0,70 % tiem 100,00:t 0% V.parahaemolyticus nj!;m 66,67 :t 6,67% tiem 100,00:t 0% V. alginolyticus nJ!.m 46,70:t 14,53% tiem 73,33 :t 3,33 % V. anguillarum ndm 30,00:t 11,54 tiem 30,OO:t5,77% V. vulnificus ngm 13,83:t 8,82% tiem 16,70:t 5,77% D6i chang ngiim 0 tiem 0 8 4.1.4. Nghi~n cl1u chi t4o khang huyit thanh V. harveyl dg phat hi,n nhanh vi khu§n nay tr6n tom Thi nghi~m du'<Jcthl1c hi~n v~i khang nguyen la chUng V. harveyi du'<Jcphan l~p, xac nh~n tinh gay b~nh va du'<Jclam nhu'<JcdQc.Khang nguyen nay du'<Jcdung d~ gay dap 1i'ngmi~n dich tren ca tai tu'<Jng (Osphronemus gourami). Khang huytt thanh du'<Jcdung d~ chin doan nhanh V. harveyi . Ktt qua nhu'san: (Bang 4.1.4). Bang 4.1.4 Hi~ngiakhangthl eua Vibrioharveyic6 tronghuy€tthanheua ea tai t1Jqnggiii'acae 16ea thi nghi~m Ghi eM: +,++,+++: tM hi~n mile dq ngltng Mt; (-J: khOng xudt hi~n ngltng Mt Hlnh 4.5. PhaiRang ngungk't giO'akhang nguy6n (V. harveyi) Ydikhang huy't thanh cua V. harvey/ (X 10) (a): (leSichang am; (b): khang huy't thanh phs lo8ng 1:5 (c): khang huy't thanh phs lo8ng 1:10 DQpha Vitri nerokMnonolNen loting Tinh ch dui Xoan2 b\ll12 huy't Mi\u 1 Mi\u 2 Mi\u 3 Mi\u 4 Mu5 Mu6 thanh 1 : 5 +++ +++ +++ +++ +++ +++ 1: 10 +++ +++ +++ +++ +++ +++ 1: 20 ++ ++ ++ ++ ++ ++ 1 :40 ++ ++ ++ ++ ++ ++ 1 :80 + + + + + + 1: 160 + + + + + + 1: 320 + + + + + + 1 :640 (-) (-) (-) (-) (-) (-) [...]... LU~N CHUNG: 1 Mt1m ~nh vi sinh hi~n di~n tren tom su (P monodon) nuoi (j BBSCL baa g6m ca vi khu.1n (nh6m Vibrio) va virus (MBV, WSSV) Chung c6 th~ xu!t hi~n tren cac mo hinh nuoi: QC, BTC, "Tom -Lua" va nuoi th . Mn vung (j Vi~ t Nam, ct,lth~ la cac dnh D6ng B?ingS6ng Cii'uLong (DBSCL). 1.2. Ml,lcti~u cua luin an D~ tai du'<;1cthf!Chi~n nhiim xac djnh vi sinh v4t (vi khua'n va virus) gay. eU'u~nh vi sinh v~t (virus va vi khu~n) tren tom so.va cac loai tom he tren the' gidi va aVi~t Nam. Chlidng 3: V4T lieU vA PHUONG PHAp NGHIEN CUU 3.1. Nghi6n cuu mSm btnh vi khuin tr6n. va phu'dngsai ANOVAde timcacm6itu'dngquaD. 3.2. Nghi6n ct1u mim b~nh virus: virus g6y b~nh col (MBV)va virus g6y hoi chang d6m trAng (WSSV)tr6n t6m su va cae loai is hQt6m he 3.2.1.