KHOA HQC CdNG NGHE
DAIMH GIA HIEU QUA UC CHE VI KHUAIV VitBria pmrmhmemaiyticus BOB 1.4M/
EiAY BEI\IH TREIXI TOIVI CUA Baciiius suistiiis
U Thd XuanS Pham Anh Tu^[n^ Vu Ngpc Ut^
T6MTAT
Nghifin cmi duoc thuc hi&n nham danh gia hieu qua iic che vi khuan Vibno parahaemolyticus gay benh hoai tCr gan tuy d t6m ciia cac dong vi khuan Bacdlus BRB2.1 va BDK2.3 phan lap tir khu vuc nuoi t6m d Bac Lieu. Nghien cmi gom 2 thi nghiem. O thi nghiem 1, torn the chan trdng duoc bd tri trong be 100 Ht vdi mat do 30 con/be. Thi nghiem 2 duoc thiet ke trong be composit 1000 lit trong do mat dp torn la 200 con/be va vdi dp man la 20%o, thdi gian nuoi la 35 ngay. Trong ca 2 thi nghiem, torn ddu diroc gay cam nhifem vi khuan V. parahaemolyticus-vtn co bd sung va kh6ng bd sung vi khuan Bacillus B^2.1 va BDK2.3. Mpt sd chi ti^u chat lirong nuoc, tdng vi khuan, vi khudn Vibrio duoc theo doi 3 lan/ngay, Kdt qua cho th^y, sau 35 ngay, ty le sdng cua tdm b nghiem thiic bd sung vi khuan BRB2.1 dat cao nhat (87,83%) va khac biet co y nghla (p<0,05) so vdi cac nghiem thiic con lai. So vdi dong BDK2.3, dong BRB2,1 co hieu qua tdt hon dua frSn cac chi tieu ve smh tmdng va ti 1§ sdng cua tom. Do do, dong Bacillus BRB2.1 duoc xem la ddng vi khuan co loi, co the uc che su phat trien cQa V. parahaemolyticus.
TiiViida: Baa'ilus subtilis. Vibrio parahaemolyticus, die torn sdng sot, b€nh hoai td gan tuy cap, ikchd.
1. BAT VAN OE
Hien nay Hpi chiing hoai tii gan tuy cap tinh (AHPND) gay henh chet sdm tren torn the chan trang da dupe xac dinh la do vi khuan Vibrio parahaemolyticus (Loc Tran et al, 2013). Day Id 1 trong cde dich benh gay ehdt, lam giam san lupng [6m nudi ddng kd d cac nude trong khu vuc Ddng Nam A 0 Viet Nam, AHPND dupe phat hien tii nam 2010 nhung miic dd Ihiet hai dupe ghi nhan nhidu nhdtvdo nam 2011 va 2012. Dien tich nuoi tdm hi anh hudng bdi AHPND d dong hdng sdng Cuu Long (DBSCL) khoang 39.000 ha ti-ong nam 2011 va 2012 (FAO,2013).
Dd han ehe thiet hai, khdng sinh, hda chat da dirpc su dung kha phd bidn dd phdng vd tri bdnh va dieu do dan den nguy co tao ra nhirng dong vi khuan khang thudc nguy hiem. Nhieu nghifin ciru da cho thay c6 thd ban ehe khang sinh bang each su dung mpt sd ddng vi khudn cd lpi dd kidm soat vi khuan gay b6nh trong qua trinh nudi tdm va day eo the dupe xem la giai phap hiru hieu trong phdng, tri hpi chiing chet sdm d tdm the chan trdng. Trong eac nhom vi sinh vat eo lpi duoe su dung pho bien hien
nay. Bacillus spp. luon Id thdnh phdn chii ydu trong cac san pham ehe pham sinh hpc, do chiing cd kha nang phan huy eac chat hdu ca, lam giam khi doe, cai thifin chat lupng nude cung nhu ed kha nang lie ehe eac vi khuan cd hai. Nhieu nghi6n ciiu da dupe thuc hien vdi viec sii dung cac dong Bacillus dd ban chd vi khudn Vibrio parahaemolyticus gdy henh trong ao nuoi. Wang et al. (2006) sii dung ehe pham sinh hpc vdi thdnh phdn chinh Id Bacillus sp. trong ao nudi tdm the chan trdng da han ehe duoc vi khudn Vibrio. Trong mdt nghifin ciiu khae, khi bd sung Bacillus sp. vdo mdi trudng nuoi tdm trong dieu kien phdng thi nghiem, chat lupng nude dupe cai thien vd mat dp Vibrio giam dang ke (Pham Thi Tuyet Ngan, 2011). Tuong tu nhu vay, Vaseeharan vd Ramasamy (2003) cung ket luan rdng Bacillus trong didu ki§n phdng thi nghiem vd ngoai thue te cd the Idem soat dupe mam h6nh Vibrio. Tbeo Stein et al. (2004) vd Babasaki et al. (1985), Bacillus subtilis ed the san xuat subtilosin A - mot trong so peptid khang khuan CO kha nang lie chd cae nhdm Vibrio gay henh. Muc tieu ciia nghien ciiu nay la tim ra dong vi khuan Bacillus cd kha nang ban chd su phdt trien eua Vibrio parahaemolyticus gay benh hoai tir gan tuy d torn nudi trong ao.
Nghien ciiu sinh, Khoa Thiiy san, Tnrdng Dai hoc Can Tha
Hoi Nghe ca Viet Nam
^ Khoa Thiiy san, Tnrong Dai hoc CSn Tho
KHOA HQC CONG N G H £
2 . VfIT LEU VA PHUDNG PHAP lUeHBU CUU 2.1. v a t h ^ nghidn ciiu
Nguon vi khuan: Cae ddng Bacillus sii dung trong nghien ciiu nay dupe phan lap tir 27 mdu nude, 27 mdu hiin vd 18 mau tdm trong 10 ao tom benh vd 10 ao tom khoe tai 3 tinh Bac Lieu, Cd Mau va Sdc Trang. Tdng sd 126 chtmg Bacillus da dupe phan lap trong do 30 ehung dupe chpn dd thi nghifim dua vdo dae diem hinh thdi, smh hoa va phan tich trinh tu IBS rRNA.
Tuong tu nhu vay, cae chung vi khuan Vibrio cdng dupe phan lap tu nude, bim va tdm trong cde ao tdm d Soc Trang, Bac Lieu va Cd Mau. Trong so cde ehung ndy, 30 chung dupe sang loc sau khi nghien ciiu gen ddc c6 kha nang gay benh nguy hidm va gay chet tdm nudi. Trong 30 chung phan lap duoc, BDB1.4v la chiing dupe chon thudc loai V.
parahaemolyticus cd khudn lae mau tim tren mdi trudng CHROMagar. Vibrio duong tinh vdi gen VpuA, VpirB, toxR vd toxR-S vd am tinh vdi gen tdh va trh dupe su dung de tidn hanh thi nghiem,
Cac chung vi khuan da chon dupe nudi tang suih trfin mdi trudng LB. Sau khi tang sinh, mat dp vi khuan duoe xac dinh bang phuong phap do OD d bude sdng 600 nm trude khi cay chung vdo cae nghi6m thiic thi nghiem.
2.2. Phuong phdp nghito ciiu
2.2.1. Tuyen chpn chiing Bacillus sp. co hoat ti'nh ddi khang Vibrio parahaemolyticus
Hoat tinh khang khudn eiia 30 chung Bacillus ddi vdi chiing V. parahaemolyticus BDB1.4v duoc xac dinh bdng phuong phap khuech tan tren thach.
0,01 mL dich huyen phii vi sinh vat Idem dmh (cde chiing Vibrio sp.) dupe nhd len bd mat mdi trudng thach vd dan deu hang que cdy. Cac 16 tren dia thach duoe due hdng khoan nut. Sau do, 0,1 mL dich huydn phii vi khuan Bacillus sp. da nuoi d moi trudng LB long dupe cho vao cac 16 thach va nuoi trong tu am 30°C. Sau 24h - 36h cde dia thach nay dupe Idem tra vdng uc che vi sinh vat kiem dinh (D-d mm) dd xac dinh hoat tinh khang khuan (Nguydn Lan Dung vd ctv, 2012).
2.2.2. Kha nang uc che Vibrio parahaemolyticus ciia cac chiing Bacillus duoc lira chon
Tu ket qua danh gia tinh khang khudn 2 ddng Bacillus da dupe chon de thuc hien thi nghiem la
BRB2.1 va BDK2.3. Kha nang lie chd K parahaemolyticus ciia cde chung Bacillus sp. dupe thue hi6n qua 5 nghiem thiic, mdi nghiem thiic dupe lap lai 3 lan, bao gom:
NT Al: Ddi ehung (khdng hd sung vi khuan Bacillus va V. parahaemolyticus)
NT A2: V. parahaemolyticus BDB1.4v (10^
chi/mL)
NT A3: Bacillus BRB2.1 (10^ cfu/mL) + V.
parahaemolyticusBBBl.iv (10'' chi/mL) NT A4: Bacillus BDK2.3 (10^ cfu/mL) -t- V.
parahaemolyticusBBBl.Av (10^ cfu/mL)
NT A5: Bacillus (BRB2.1 -H BDK2.3) (10^
chi/mL) -H V. parahaemolyticus BDB1.4v (10^
chi/mL)
Thi nghiem dupe bo \ri frong 15 bd vol the tich 100 L nude cd dd man 20%o (da xii ly chlorine 30 ppm) dupe sue khi. Tdm the chan ti-dng, giai doan postlarvae (PL 15 • 20), sach benh da dupe xet nghiem cac tac nhan gay henh (WSSV (White-spot syndrome virus), IHHNV (Infectious Hypothermal and Hematopoietic Necrosis Virus), AHPND (Acute hepatopancreatic necrosis disease), EHP (Enterocytozoon Hepatopenaei))... bdng phuong phap PCR vd phan tich mo, duoc bd tri vdi so lupng 30 ed the/be.
Hai chtmg vi khuan Bacillus dupe nudi tang sinh trong moi trudng LB trong thdi gian 24 gid. Sau do mat dp cae chung vi khuan Bacillus dupe xac dinh va cay cde vi khuan Bacillus ndy vao thung chiia 30 con tdm sach benh {da dupe xet nghiem) vdi mat d6 10^
chi/mL.
Chiing vf khuan V. parahaemolyticus BDB1.4v duoc nudi tang sinh trong moi trudng LB trong 24 gid. Mat dp vi khuan sau dd dupe do vd cdy vao mfli nghiem thiic da dupe cay chiing vi khuan BacHlusvm mat dd lO'' chi/mL.
Bidu hien md benh hpc vd ti le song eiia torn duoc theo ddi vao cac thdi diem sau 12, 24, 36 va 48 gid thi nghiem.
2.2.3. Kha nang khang Vibrio parahaemolyticus ciia cac chung Bacillus
Thi nghiem gdm 3 nghiem thiic, mdi nghiem thiic dupe lap lai 3 Idn, hao gdm:
NT BI: Nghifem thuc ddi chimg (khdng cay vi khuan).
NT B2: Chi cdy Vibnoparahaemolyticuswbi m^t
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2019
KHOA HOC CONG NGHE
do 10* chi/mL
NT B3: Cay Bacillus BRB2.1 (10^ cfu/mL) -H Vibrio parahaemolyticus (lO^chi/mL)
Thi nghiem dupe tidn hanh trong eac he composite dung tich 1,2 m^ dat trong he thdng nha ludi. Nude bien co dp man 20%o dupe xu ly bang elorine (30 ppm) va sue khi h^n tuc cho den khi het du lupng elorine, sau dd dupe cap vao be qua tiii Ipe (Spm).
Tom the chan trdng cd kich cd trung binh 38- 40 g/con dupe xet nghifem eac tac nhan gay benh bdng phuong phap PCR vd phan tich mo de lua ehpn tdm gidng sach benh dua vao thi nghiem. Tdm dupe bd tri vdo bd composite vdi mat dp 200 ca thd/be.
Tdm dupe cho an bang thire an DE HEUS 4 lan/ngdy vao cde mdc thdi gian 7g, lOg, 14g vd 17g.
Lirpng thiic an dupe tinh theo phdn tram khdi luong than va qua quan sat hang ngay de dieu chmh cho thieh hpp theo timg hd. Bd nudi dupe xi phdng 3 ngdy/lan, va duoc thay 30% nude khi cac chi ti6u mdi trudng vupt qua cae ehi ti^u cho phep.
Cae chi tieu nhidt dp, pH, oxy hoa tan (DO) dupe do dmh ky hdng ngay bdng may do didn tir;
NH3, NO2 dupe do 3 ngay/lan.
Cde chi tieu vi khuan dupe thu mdu 3 ngdy/lan vd dupe xde dinh bang phuong phdp dem khuan lac tren cae mdi trudng tiiong ling vdi tiing chi tifiu:
Vibrio hen mdi trudng TCBS va tdng khuan hen mdi trudng NA
Tdc dp tang trudng ve chieu ddi, khdi lupng, ti 16 sdng, he sd chuyen doi thiic an ciia tom dupe xdc dinh sau 15 ngay vd khi kdt thuc thi nghiem.
Tmh ti le sdng cua tdm: Ti le sdng (%) = (So ca thd cu6i/Sd cd thd dau) x 100.
Khdi luong gia tang = khdi lupng cudi - khoi lupng dau.
FCR (he so chuyen ddi thiie an) = thiie an su dung/khdi lupng tdm gia tang.
2.2.4. Phmmg phap mo hpc
Tom postlarvae dupe ed dinh bdng dung dich Davidson's hong vdng 12 ddn 24 gid, sau do dupe chuyen sang con 70" dd bao quan lau dai theo phuong phap eiia Lightiier (1996).
Mdu sau khi cd dinh dupe xii ly. Qud trinh xtr ly mdu gom loai nude, lam trong mdu va tam paraffin.
Sau dd, khudn nhua chiia mdu duoc chuydn sang
may due khdi va cdt Idt. Cae lat cdt nay dupe cho vao nude 0 nhiet dp 45-50°C lam cho parafin cang ra vd dan len lame. Lat cdt dupe nhudm bdng phuong phap nhudm Haematoxylm vd Eosin (H&E). Mdu dupe quan sat vd ehup anh dudi kinh hien vi d cae vat kinh 10X,40Xvdl00X.
Khi te hao gan tiiy tdm hi AHPND se the hien dau hieu tap trung nhidu te bdo hdng cdu, ed su hong trdc te bao trong cac dng gan va hinh thanh edc thd granuloma.
2.2.5. Nghidn cdu Bacteriocin
Quy trinh thu nhan va tinh sach bacteriocin Tang sinh vi sinh vat: Lay 1 dau que cay vi sinh vat tir dng nghiem, nudi B. subtilis BDK 2.3 vd BRB 2.1 trong mdi trudng BHI trong 18 gid.
Nuoi thu boat tinh baeteriocm trong mdi trudng BHI bo sung 1% mdi trudng BHI: ti le cap giong 10%, tong the tich nudi 30 ml, thdi gian nuoi thu baeteriocm 50 gid.
Ly tam thu dich noi (6000 rpm, 1 gid, 4°C).
Ket ttia bacteriocin trong dieu kien lanh lO^C bdng amoni sulfat 80% dp hao hda (can 16,24 g (NH4)2S04 cho thd tich 30 ml d dieu kien lO^C), u qua d6m d 4''C.
Ly tam (6.000 x g, 1 gid) thu tua va hoa tan tua ti-ong 4 ml dem A lanh (Dem A 20 mM Tns-HCl, pH7.0).
- Loai mudi bang mang tham tich 2000K (Shde-A-Lyzer 66230 Thermo scientihc) trong ddm A, thdi gian loai trong 24 gid, d nhifet dp IS^C.
Loc qua cot cut off lOkDa, lay dich dudi cot.
Tua bdng edn 70°C, hda tan lai trong 200 jil dem A
Sau khi bacteriocin dupe thu vd tinh sach, xac dinh boat tinh uc chd vi khuan eua no bdng phuong phdp da dupe mo td (AU/mL) (Hashium etal., 2010).
Hoat tinh bacteriocin dupe thd hien thdng qua nhidu don vi khac nhau nhu: AU/mL (arbitrary unit), BU (baeteriocm unit),....M6i loai don vi lai co mpt cdch xac dinh khae nhau. Hoat tinh bactenocin dupe the hien bdng AU/mL (activity unit) la nghich dao eiia ndng dp pha lodng Idn nhat trong day pha loang h6n tuc 1/2 ma tai do vdn ire che chung kiem dinh.
AU/mL dupe tmh bang cdng thiic:
AU/mL = Vv x 2 " x l 0 0 0 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2019
KHOA HOC CONG NGHE Trong dd: V: la lupng the tich dich pha loang hiit
vao mdi gieng de thu hoat tinh (uD; 2": la dp pha loang thii n ma cd the hien hoat tinh baeteriocm.
Tach ddng gen ma hda baeteriocm tii chiing Bacillus subtihs (gen suhtilosm-^ hdng cap moi dupe lua chpn cd trinh ttr
Subtilosm-F: ATGAAAAAAGCTGTCATTGTAGAAA a m = 62).
Subtilosm-R: TTATCCCCATAGACCGAATAGAC a m - 6 3 ) .
Toi uu hda phan ling PCR khudeh dai gen ma hda suhtilosm-A (SBO) hdng each sii dung nhidt dp bdt cap moi d 54"C, 56°C, 58°C va 60°C.
2.2.6. Phuong phap xdly so heu
Sd heu thu dtrpe d cde thi nghiem dupe ti'nh gid tri tnmg hinh va phan tich phuong sai mpt yeu to (ANOVA) d miic khae biet P<0,05 hdng phdn mdm thdngke SPSS 16.0.
3. KET QUA VA THAO L U M
3.1. Tuydn chpn chung BacHhis ^ . cd hoat tinh ddi khdng Vibrio parabaemofytictis
Ket qua nghien ciiu tinh khang khuan cua eac chiing Bacillus dupe the hien d bang 1 va hinh 1.
Trong tdng sd 30 chiing vi khuan Bacillus dupe chpn, chi cd 6 chiing cd xuat hien vdng khang khuan khi thuc hien thi nghiem. Dudng kinh vong boat ti'nh ciia vi khuan Bacillus BRB2.1 ddi khang vdi vi khuan Vibrio BDB1.4v cao nhat la 1333 mm va khac biet cd y nghla thdng ke (P<0,05) so vdi cac chung vi khuan Bacdlus cdn lai. Chung vi khuan Bacillus BDK2.3 ciing cho ket qua tuong ddi cao la 12 mm. Hai chung Bacillus BRB2.1 va BDK2.3 nay dupe chpn de thuc hien cac thi nghiem tidp theo.
Bang 1, Vdng lic d i ^ vi smh vSt tii ki&i dmh (I><0 dia m$t s6 chiing fEac^^vdi ^brio
parabaemofyticus STT
1 Chung Bmdllus BRB2.1
Ehidng kinh vdng khang khuan (mm)
13.33+0,58^
2 3 4 5 6
BDK2.3 BNK6.1 ,
BRK4.4 BNK7.1 1 BDBll.l 1
U.OCM.OCP 5,00+l,00' 6,00±0.0CI' 8,0Q±0,00=
433±0.58' Gia tri the hien la cac gia tri trung binh ± do lech chuRn. Gia tri trong cung mot bang m mang chd cai kbacnhau thi khac biet coy nghia thdngke (p<0,(^.
Hinh 1. Vi khuan Bacillus cd hoat tinh ddi khang 3.2. Kha nang khdng Vibrio cua cac chung BacUhis
Ty le sdng cua tdm cao nhat dat 97,78% ghi nhan dupe d nghiem fiiiie Al (nghiem thiie doi chiing - khdng CO Vibrio gay benh), trong khi do d nghiem thiie A2 (ehi ed Vibrio gay henh), ti le sdng la 0% sau 36 gid. 11 le sdng ciia tdm trong cae nghiem thiic cd bo sung Bacillus kha cao, nhat la trong nghiem thiic A3, sii dung ddng Bacillus BRB2.1 (Bang 2) mac dii CO ti 16 song thap hon d nghiem thiic ddi chung. Dieu nay chung td vi khuan Bacillus trong cac nghiem
vi khiian Vibrio BDB1.4v bang phuong p h ^ due 16 thiie nay da lic ehe su phat trien cua Vibrio gay h ^ . Hasting va Nealson (1981) cho rdng Bacillus ed the tao ra mdt so chat khang khuan hoac mpt vai san pham cd the diet khuan. Tuy nhien, trong nghiem diiic A5 khi cay cung liic 2 chiing vi Idiuan Bacillus, ti le sdng ciia tdm thap hon dang ke (P<0,05) so vol nghiem thuc A3 va A4 (cay timg chiing rieng le vi khuan BacUlu^. Xhu vay, mSc du 2 ddng vi khuan Bacdlus cd the lie che vi khuan Vibrio BDB1.4v nhung khi sii dung dudi dang hiep luc thi kdt qua khdng tdt bdng su dung rieng le.
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2019
KHOA HOC C 6 N G N G H £
Bang 2. T! 1$ s6ng ciia tdm a cde nghidm thtic sau 3 6 g i d t i i i n g h i ^
Al A2 A3
A4
A5
Nghiem thiic BC BDB1.4V BRB2.1- BDB1.4V BDK2.3- BDB1.4V BRB2.1- BDK2.3- BBB1.4V
Ti 16 s6ng (%) Ciia torn 97,78±3,85' 0,00±0,00«
85,56+1,93'
76,67±6,67'
65,67+5,13''
Ket qua m6 hoc AHPNS Am tinh Dirong tinh
Am tinh
Am tinh
Am tinh
Gia tii thehiSn la cac gia tri trung binh ± do lech chuan. Gia tri trong cung mdt hang co mang chd cai kbacnhau thi khac biet co y nghia thdngke (p<0,05).
Sau khi da tach ddng gen ma hda Bacteriocin tir 2 chiing Bacillus BRB 2.1 va BDK 2.3, tien hdnh tdi uu hda phan ling PCR khuech dai gen ma hda subtilosin-A (SBO), ket qua dupe thd hien d hinh 2.
Hmh 2. Kdt qua didn di do khudch dai gen ma hda subtilosin-A
Ghi chu: dudng chay 1, 2, 3, 4 lan luat la san pham PCRkhudch dai doan Subtilosin tuchungBRB 2.1 & cac nhiet do bat cap tuong ung 54, 56, 58, dtPC;
dudng chay 5, 6, 7, 8 lan luot la san pham PCR khuech dai doan Subtilosin tu chung BDK 2.3 o cac
nhiSt dp bdt cap tirong img 54, 56, 58, &PC; dudng chay 9: mdu am tinh PCR; dudng chay 10: thang ADN Ladder (Norgen).
Chung BRB2.1 cd bacteriocin cao, cde phan ling PCR vdd cap mdi khuech dai subtilosin cho kdt qua duong tmh d ca 4 nhiet dp gan moi thii nghiem vdi kich thudc eua san pham phu hpp vdi tinh toan ly thuydt khoang 130 bp. Subtilosin A ed hoat tinh khdng khudn rpng ddi vdi ca vi khuan Gram am va Gram duong (Shelbume et al, 2007). Co chd boat dong cua subtilin dua tren khd nang tuong tac vdi cac hpp phan cua mang ngodi hpid cua td hdo vi khuan tii d6 tao ra cae 16 ti-en mang td hao kdt qua Id cde thdnh phdn quan trpng trong npi bdo se hi mat mat vdo mdi trudng vd te bdo vi khuan se hi tieu diet (Parisot etal, 2008).
Kdt qua md benh hoc cua tdm (Hmh 3) cho thdy d nghiem thuc A2 ehi bo sung vi khuan Vibrio khdng hd sung vi khuan Bacillus cho mdu duong tinh vdi benh AHPNS, d cac nghiem thiic ed cay vi khudn Bacillus vd nghiem thiie ddi chiing deu cho mdu am tinh vdi benh AHPNS. Theo Lighner et al (2012), tdm b6nh thudng cd mot sd dae diem mo henh hpc dae tnmg nhu: Thoai hod cap tinh ciia cac dng gan tiiy vdi su rdi loan chiic nang cua te bdo va nhan te bao truong to, te hdo hi hoai tu roi vao trong Idng dng gan tuy. Ngbien ciiu eiia Nguydn Trpng Nghla vd Dang Thi Hodng Oanh (2014) cho thay vi khuan Vibrio parahaemolyticus ed kha nang gay hoai tii gan tiiy cap khi gay cam nhidm tren tdm the chan trdng hang phuong phdp tiem. Loc Tran et al. (2013) cung da xac dinh dupe kha nang gay henh gan tuy cdp ciia vi khuan Vibrio parahaemolyticus tren tdm the chan trdng bdng phuong phap ngam.
Hmh 3. Td bdo gan tuy tdm hi hdi chiing hoai hi gan tuy cdp tinh (AHPNS)
Ghi chii: Mui ten mau xanh duong: Cac the granulom; Mui ten mau vang: Bong troc cac te bao trong
NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2019
KHOA HOC CONG NGHE
3.3. Kha nang lic chd cua BadBas sp. ddi voi Vibrio parahaemolyticus vd anh hudng Ign su sinh trudng ciia tdm the chan trdng
3.3.1. Cac yeu td moi trudng
Cde yeu td eua mdi tnrdng thi nghiem dupe neu d hang 3. Kdt qua cho thdy nhiet dp, pH va ham lupng oxy hda tan trung binh giiia cae nghiem thiic khong cd su sai khac Idn do cac nghiem thiic duoe hd tri trong cimg mdt khu thi nghiem. Theo Boyd (1998) nhiet dp nude thich hpp cho cde loai thuy san nhiet ddi la 25 - 32°C. Nhiet dp moi trudng trong thdi gian thi nghiem dao ddng trong khoang cho phep 29,87 - 30,2"'C. Yen td pH ti-ong qua trinh thi nghiem tucmg ddi dn dinh, su chenh lech giira cde nghiem thiic la rat nhd (8,01 - 8,04), khoang pH thich hpp cho ddng vat thiiy san Id 6 - 9 (Boyd, 1998). Trong didu kien thi nghiem, edc be nudi duoe sue khi Hen tuc nen hdm lupng oxy hoa tan trong nude tuong ddi cao va dn dinh, ham lupng oxy dao ddng 8,75 - 8,94 mg/L. Ciing theo Boyd (1998), ham lupng oxy hda tan phu hpp cho thuy smh vat la 5 -15 mg/L. Vi vay, nhidt dp, pH vd ham lupng oxy hda tan trong didu kien thi nghiem phu hpp cho su phdt trien binh thudng cua tdm the chan trdng.
Bang 3. C^ chi tidu mdi trudng trong cac n g h i ^ tfaiic
lupng XH, phai nhd hon 0,1 mg/L va ham lupng N0_
khong nen vupt qua 10 nig/L, tdt nhdt la nhd hon 2 mg/L (Boyd. 1998). Nhuvay, ham lupng XH^va .NO, do dupe trong cac nghiem thuc ddu ndm trong khoang thich hpp cho tdm the chan trdng.
3.3.Z Vi khuan
Mat dp vi khuan tdng sd (tdng khuaiO d cac nghiem thiie eo hd sung vi khuan dao ddng trong khoang 10^ - 10^ CFU/mL va khac hi&t cd y nghia thdng ke (p<0,05) vdi nghiem thuc doi chiing {W - 100 CFU/mL Rieng doi vdi nghiem thiic ddi chiing, mat dd tdng khuan tang ddn theo thdi gian do su tich luy vat chat hiiu co do thiic an va chat thai cua tdm.
Sau 31 ngay nudi, mat dp tong khudn d nghiem thuc B3 cao nhat 1,3.10^ CFU/mL (Bang4).
Bang 4. Mat dp vi khuan tdng sd dia c ^ nghidm thiic (CFU/mL)
Chi tieu
^^hietdp (t°C) pH DO (mg/L) NHj (mg/L) NO, (mg/L)
BI 29,77±0,01
8,04±0.01 8,94+0.03 0,11±0.02 0,30+0.07
B2 30,23+0,27
8,02+0,03 8,90+0.05 0.09±0,01 0.10+0.00
B3 29.97+0,04
8,01+0.02 8.75+0.03 0,12+0.01 0.67+0,03 NH3 dao dong giira cac nghiem thiic 0,09 - 0,12 mg/L va CO xu hudng tang theo thdi gian, tuy nhien, khi NH. tang qua miie cho phep thi cac be nudi dupe thay nude de duy tri trong gidi han cho phep. Rieng nghiem thiic B2, torn ehdt 95% sau 36 h nen nghiem thiie da dupe kdt thiic vd ghi nhan ket qua. Ddi vdi cac nghiem thiic con lai thi nong dp NH3 cao nhdt d nghiem thiic B3 (0,12 ± 0,01) mg/L va thap nhdt b nghiem thiic BI (0,11 ± 0,02) mg/L, tii dd ed the thay dupe d cac nghiem thiic cd bd sung Bacillus cac chat hiiu eo trong be se dupe chuyen hda manh hon de tao thdnh NH3 thong qua qua trinh amon hoa. Ham
Ngay nuoi 0 4 7 10 16 23 27 31
B1(BC) 1.110=
l,7.1ff"' 8,0.10=' 1,3.10"
1,9.10"
3,0.10*' 5,7.10"
4,1.10"
B2 1,9.10*
1,3.10«"
- - - - -
B3 1,6.10=
l.O.lOe- 9,3.10=»
l.e.lff^"
2,1.10«*
1,1.10»"
2 3 . 1 0 "
1,3.10"
Gia tri the hien la cac gia tri tnmg bind ± dp lech chuan. Gia tri trong ding mot hanged mang chu cai khacnhau thi khac biet coy nghia thdngke OKO,05).
Mat dp vi khudn Vibrio sau 4 ngay thi nghiem cao nhat 0 nghiem thiic B2 (5.10^ CFU/mL) va khac biet cd y nghia so vdi eac nghiem thiic khde. Day la nghiem thiic dupe cdy vi khuan Vibrio paraheamolyticus nhung khdng cay \ i khuan Bacillus. Tdm trong nghiem thiic nay chet den 95%
sau 36 gid. Theo Moriar^ (1999) mat dp vi khuan VibriovMOi qua 10^ CFU/mL se g ^ hai cho tdm. Mat dp Vibrio a nghiem thtic ddi chiing thdp nhdt (1,3.10^
CFU/mL) do khdng cdy vi khuan Vibrio.
Sau 7 ngay thi nghiem mat dp Vibrio a nghiem thiic ddi chung BI vdn t h ^ nhat nhung ed xu hudng tang len ve cudi thdi gian thi nghiem (Bang 5).
Trong khi dd, mat dp Vibrio a nghiem thiic co Bacdlus (B3) giam manh sau 7 ngay nudi khde biet
84 NONG NGHIEP VA PHAT TRIEN NONG THON - KY 1 - THANG 1/2019
KHOA HOC C 6 N G NGHE
cd y nghia so vdi nghi§m thiie eon lai. Didu d6 cho thay viec bo sung vi khudn Bacillus da cd tdc dung at chd vi khuan Vibrio paraheamolyticus. Vi khudn Bacillus cd thd ddng vai trd nhu mot tdc nhan smh hpc gilip lam giam Vibrio trong he thong nudi thuy san (Rengpipat, 1998), tuy nhidn d nghiem thiic doi chiing lai eo xu hudng tang do su tich luy ehdt huu eo trong be nuoi.
Bang 5. M^t dp vi khudn W&nro d cde nghidm thiic thinghi|Sm
Ngay nu6i 0 4 7 10 16 23 27 31
B1(BC) 1,5.10^
1,3.10"
2,3.10"
2,9.10"
2,8.10"
5,4.10"
7,5.10"
1,5.10*
B2 1,8.10*
5,0.10"
B3 1,6.10' 2,8.10"
1,3.10"
l.l.lO"-' 7,5.10"
5,1.10"
4,3.1ff"' 2,7.10"
Gia tri thehi0n la cac gia tri tnmg binh ± dp lech chuin. Gia tri trong cimg mpt hang co mang chu cai khacnhau thi khac bi$t co y nghia thdngke (p<0,05).
3.3.3. Tyle sdng va tang trudng ciia tdm T\ te sdng eiia tom the chan trdng d eac nghiem thiie thi nghifem dupe trinh bdy d hang 6. Trong nghiem thiie B2 (ehi ed V. parahaemolyticus, tdm chet 95% sau 36 gid. Trong edc nghiem thuc edn lai, tom d nghiem thiie B3 (bo sung Bacillus) cd ti 16 sdng cao nhdt (87,83%). Mac dii, torn trong nghiem thiic B3 gay cam nhidm vdi vi khuan Vibrio parahaemolyticus nhung dong tihdi ed bd sung Bacillus BRB2.1 nen tom trong nghiem thiic ndy vdn phdt tnen tdt vdi ti le sdng cao nhat. Ket qud ndy hodn toan phii hpp vdi nghien ciiu ciia Pham Thi Tuydt Ngan va Trdn Suong Ngpc (2014) cho thdy viec bo sung vi khuan Bacillus sp. se cai thien ti le sdng cua tdm the chan trang. Vi khuan Bacillus cd kha nang sinh ra 24 loai khdng smh khae nhau dudi cac dang nhu polypeptide, hpopeptide, polyketide, anunosugar, phospholipid...(Stem etal, 2005), trong do Lipopeptide Id chat hoat ddng he mat giiip cho vi khuan Bacillus ed thd tiep xiie vdi td bdo vi khuan Vibrio va gay iic chd chung (Stein et al, 2005, Versehuere et al, 2000). Theo Vaseeharan et al (2003) su dung vi khudn Bacillus trong ao nuoi torn
gilip tang cudng su tang trudng vd nang cao ti le sdng eua tom.
Mac du thdi gian thi nghifem tuong doi ngan (35 ngay) nhung he sd chuyen doi thiie an (FCR) cua tdm thi nghiem cung co su khae bidt giiia cac nghiem thiic. He so chuyen doi thiic an d nghiem thiie B3 la thdp nhat (1,11) va khde biet ed y nghia thong ke (p<0,05) so vdi cde nghiem thiic doi chiing BI. Didu d6 cho thay viec bd sung vi khuan Bacillus BRB2.1 ngoai vi6c lie che V parahaemolyticus, dong vi khuan co lpi nay cung da gdp phan vdo viec tang hieu qua hap thu vd chuyen hda thiic an ciia torn the chan trdng. Theo Ziaei-Nejad (2006) vi khuan hiru ich ed thd kich thich cde enzyme npi sinh lam tang hoat ddng cua cac enzyme tieu hoa ddn den tang cudng he Ueu hda va tang kha nang hap thu thiic an cho tdm.
Bang 6. TI Id sdng cua tdm the chan trdng sau 35 ngay thi nghifem Nghiem thiic
Ti le sdng (%) FCR
BI (DC) 55,00±3,97>
1,32±0,03'
B2 B3
87,83+4,07' 1,11+0,05' Gia tri thShiin la cac gia di trung binh ± do lech chuin. Gia tri trong cimg mot hang co mang chd cai khacnhau thi khac biet coy nghia thdngke (p<0,05).
4. KETLUAN
Chiing vi khudn Bacillus BRB2.1 phan lap tii viing nudi tdm d ddng hdng song Cuu Long cd the lie che duoe Vibrio parahaemolyticus \2.c nhan gay henh hoai hi gan tuy cdp d tdm nuoi, tang ty le song eua tdm va nang cao hieu qua su dung thiic an trong nuoi tdm.
TAI UEU THAM KHAO
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KHOA HOC C O N G NGHl
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Lightner, 2013. Determmation of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp.
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KHOA HOC CONG NGHE
EVALUATION OFTHE EFFICACY IN INHIBrnNG Vibrio parahaemolyticus BDBl.dvCPiSJ^mG DISEASE IN SHRIMP OF BaciUus subtilis
Le The Xuan, Pham Anh Tuan, Vu Ngoc Ut Summary
The present study was performed to evaluate the efficiency in inhibiting growth of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) m white leg shrimp {Utopenaeus vannamei) of the Bacillus strain BRB2.1 and BDK2.3 isolated ui shrimp culture area in Bac Lieu. The study consisted of two esqieriments. The first expenraent was performed in a 100 L tank system in which shrimp were stocked at density of 30 inds/tank. In the second experiment, shrimp were set up in 1000 L composite tanks system at a density of 200 inds/tank with salinity of 20%o for 35 days. In both experiments shrimp were challenged with V. parahaemolyticus in the conditions of with and without supplemental Bacillus strain BRB2.1 and BDK2.3. Total bactena, Vibno counts and some water parameters were recorded every 3 days during the culture penod. The results indicated that after 35 days of culture, survival rate of shrimp in treatments provided BRB2.1 were highest (87.83%) and significandy higher than that of other ti-eatments and this Bacdlus stram is considered a beneficial bacteria that can mhibit growth of Vibrio parahaemolyticus.
Ifeywords: Bacdlus subtilis. Vibrio parahaemolyticus, shrimp survival, acute hepatopancreatic necrosis disease, inhibiton.
Ngudi phan hidn: PGS.TS. To Long Thdnh Ngdy nh4n bai: 02/11/2018
Ngdy thdng qua phan hidn: 4/12/2018 Ngdyduy^t dang: 11/12/2018
DtNHCHiNH
Tap chi Ndng nghiep vd PTNT sd 20, ky 2 tiiang 10 nam 2018 cd dang hdi: " NGHIEN CUU TACH DONG GEN M A H O A BACILLUS SUBTTUS TU CHUNG BACILLUS SUBTILLS PHAN LAP DUOC TU AO NUOI TOM CONG NGHIEP", tac gia: Le Anh Xuan, Khudt Hiiu Thanh, Pham Tidn Dung, Le Quang Hoa.
Trong qud trinh bien soan hai hao da ghi nham mpt sd chu trong ten hai vd ten tdc gia, nay xm dupe dpc lai nhu sau: "NGHIEN CUU T A C H DONG GEN M A H O A BACTERIOCIN TU CHUNG BACILLUS SUBTILISVUm LAP DUOC TU AO NUOI TOM CONG NGHIEP", tac gia:
Le Thd Xuan, Khuat Hiru Thanh, Pham Tien Dung, Le Quang Hda, Ph&m Anh Tudn, Vu Ngpc UL Thanh that xin Idi quy ban dpc!
Nhdm tdc gia bdi hao.
