KHOA HOC KY THUAT THO Y TAP XX SO 3 - 2013
TiNH AN T O A N V A HIEU QUA C O A VACXIN VO HOAT PHONG BENH DO VI KHUAN STBEPTOCaCCUS INIAE^kX RA TREN CA CHEM
{lATES CAlCARIfER, BLOCH 1790) NUOl TAI K H A N H HOA Nguyin Hiru Ddng, Trdn VyHich Trung tdm nghien cim gidng vd dich b^nh thuy sdn
Tnrong Dgi hgc Nha Trang T O M 1 A I
Vacxin phdng bgnh do vi khuan Streptococcus iniae gay ra tran cd chgm nudi tai Viet Nam d3 dugc thir nghiem san xudt tir chung vi khudn VN 091211R phan lap tir cd bgnh vd vo ho^it bdng formalin 0,5%. Ket qua thii nghiem in vitro vd m vivo tran cd chem cho thdy rdng cha phdm thu nghiem nay Id vd khudn, an toan va tao ddp iing mien dich ddc higu tran ca chem thi nghigm vdi he sd bao ve tucmg doi dat tir 79-86% liiy thugc vao cudng dg cdng cudng dgc. De cd tha phdt trien che phdm thir nghiem nay thdnh sdn pham thuong mai, gdp phan phat trien ben viing ngha nudi cd chem tai Viet Nam, can tiap tyc khao nghiem thdi gian bdo hg ciia vacxin va cdc bien phdp ndng cao hieu qua dap iing mian dich cua ca.
Tie khoa: Ca chem. Streptococcus iniae, Vacxin, Tinh Khanh Hda
Safety and protection potency on formalin-killed vaccine against streptococcosis In cultured baranmundi (Lates calcarifer Bloch, 1790)
In Khanh Hoa province
Nguyen Huu Dung, Tran Vy Hich
SUMMARY
A formalin-killed vaccine against streptococcosis in barramundi was experimentally produced using the bacterial strain VN091211R isolated from diseased fish in Khanh Hoa province, Vietnam.
Results of m vitro and in vivo tests showed that the vaccine is safe and produced protection effectiveness to barramundi against Streptococcus iniae infection with RPP ranging from 79-86%
depend on intensity of the challenging dose. For commercialization of the experimental vaccine, a study on protection period and measurements to improve the immune response of the fish is needed.
Key words: Barramundi, Streptococcosis, Vaccine, Khanh hoa province
I. D A T V A N D E cdn thdp. Nguyan nhan cua hien tugng nay la he qua tdng hgp cua nhidu nhan td. Tuy nhien, ciing Trong nhirng ndm qua, ngha nudi ca hian
ndi chung va ca chem {tales calcarifer Bloch, 1789) ndi ridng da phdt trian manh me tai cac tinh ven bien Viet Nam. Ngha nudi ca chem phat trian nhanh chdng nhd uu didm la loai cd
nhu cdc loai cd nudi khdc, benh truyen nhiSm ddng mgt vai trd hat sirc quan trgng, frong do, bgnh do vi khuan Streptococcus iniae gay ra la mgt trong nhirng bgnh nghiem trgng.
nay cd the chd bian phile ddng lanh da xudt khdu Vi khuan Streptococcus iniae ciing da dui?c ra thi trudng quoc ta vdi gia cao. Tuy nhian, han phan lap hr cac dgt dich benh tren ca hdi van che ciia ngha nudi ca chem hien nay la ty le song Oncorhynchus mykiss a chau Au (Ceshia et al, 62
KHOA HQC KY THUAT T H C Y TAP XX SO 3 - 2013
1992), cd cam d Nhdt Bdn (Kusuda et al, 1976) vd tran nhieu loai ca nudi, nhuyen thd vd gidp xac khdp tha gidi (Bachrach et al., 2001). Gdn day, S. iniae da dugc phan lap tir ngudi nhiam cac benh ve hoai tir ta bdo, nhiam khuan tuy sdng va nhilm khudn tdng the, tha hign mdi nguy ddi vdi sirc khde cgng ddng (Weinstein, 1997). Dieu nay cho thay S. iniae Id mgt lo^i vi khudn nguy hidm ddi vdi cd chem ciing nhu cac loai cd bian khdc.
Bgnh do vi khudn S. iniae cd tha phdng tri bdng each sir dung khdng sinh enrofloxacin (Stofiregen et al, 1996), tuy nhien phucmg thiic ndy cd tha ddn dan tdc ddng tieu cue ddi vdi mdi trudng ciing nhu tao nen cac chimg tac nhdn gdy benh khdng thudc. Vi vay, viec phdng ngira dich benh bang viec sii dyng vacxin phdng banh la mdt yeu cau cdp thiet ddi vdi tdc nhan gdy benh nay.
Bdi bdo ndy trinh bay viec thii nghiem phdng banh Streptococcosis d cd chem, bang vacxin vd hoat tii cdc chitng vi khudn Streptococcus iniae phan Idp tir ca chem benh tai Viet Nam. Viec thii nghiem nay dugc thiet ke dua tren cdc nguyen tdc eo bdn neu tai Chuyen khao dugc hgc chdu Au sd 1581: Vacxin (vd boat) phdng benh do vi khudn Vibrio gay ra tran cd hdi.
II. V A T L I E U V A PHtTOfNG P H A P N G H I E N cifu
2.1. Chung vi khuin thir nghiem vd chuan hi vacxin
Chung vi khudn S. iniae VN09I2I1R (Nguyln Hun Dung va Trdn Vy Hich, 2012) phan lap tir ca chem nudi trong he thdng muong ndi d Khanh Hda dugc sir dung lam nguyan lieu de san xuat vacxin thu nghiem.
Vi khuan gdc dugc nuoi cay tang sinh trong mdi trudng TSB bd sung 2% NaCI dat trong hi dm Idc dao (180rpm) d nhigt do 28°C trong 24 gid. Ta bdo vi khudn sau khi thu hoach dugc nia 3 lan bdng dung dich muoi sinh ly dem phosphate (PBS, pH 7.4) rdi pha loang trd lai bdng PBS da dat mat do 10' CFU/ml. Vd boat hda vi khuan
bang each them formahn vao hdn dich de dat ndng dg formalin cudi ciing bdng 0.5% va bao qudn d nhigt dg 4''C.
2.2. Kiem djnh tinh vo khudn cua che pham vacxin
Cdy hdn dich vi khuan vd boat tren cac mdi tnrdng NA, TSA, TCBS, vd KF (0,1 ml hdn djch /dia thach) rdi lid nhiet dg28"C. Sau 48 gid nudi cay, cac dTa thach dugc kiam tra da kiem dinh sir xuat hi^n ciia cac khudn lac vi khuan tran moi trudng nudi.
2.3. Kiem djnh tinh an toan doi vdi ca chem cua hon djch vi khuan vo hoat
Cd thi nghiem
Cd chem (khdi lugng thdn trung bitih II,6±1,3 g, chieu ddi than 8,3±0,4 cm) dimg trong kiem dinh nay dugc sdn xudt bdng sinh sdn nhdn tao vd ucmg nudi thdnh cd gidng tai Trung tdm nghian ciru gidng vd dich benh thiiy san - Trudng Dai hgc Nha Trang. khde manh, khdng cd ban sir cdm nhiam vi khuan S. iniae va da thich nghi vdi he nudi thi nghiem tir giai doan cd huong cho dan khi bat ddu su dyng cho hoat dgng nghien ciiu.
Tiem vacxin cho cd vdphdn biet cdc nhdm cd thi nghiem
Trudc khi tidm vacxin hodc dung dich nude mudi sinh ly, ngung cho ca dn tdi thidu 24 gid.
Khi tiam cho cd, timg dgt cd thi nghiem khodng 15 con / nhdm dugc bat ra khdi be lim giii bang vgt, gay ma cd bang dung djch Ethyl eneglycol monophenylether 100 ppm rdi tiem vacxin hoac dung dich mudi sinh ly. Ca dugc tiam vacxin vdo xoang bung (intraperitoneal injection - IP) vdi lieu lugng 0.3 ml/con, ca ddi chiing dugc tiem (IP) 0.3 ml dung dich NaCI 0.9%. Cd hem vacxin vd cd ddi chiing dugc nudi dudng trong cac ba nudi rieng biet vdi ciing diau kien be nudi vd cha do chdm sdc nhu nhau.
Dieu kien nudi diroTig cd thi nghiem Cd thi nghiem dugc nudi trong cac bd composite dung tich 2m\ chiia nude bien do man
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25-30%o, d nhigt do phdng (27°C ± 3°C). Cho cd an thiic dn vian (Seabass Feed-Uni-President Viet Nam), mdi ngdy 2 lan, cho dn ch$m rdi bdng tay cho den khi cd thdi bdt mdi, ghi chdp lugng thirc dn da sir dyng sau mdi ldn cho dn.
Quan sdt. theo doi cd thi nghiem. tdi phdn Igp VI khudn vd chdn dodn h^nh
Quan sal ca thi nghigm lidn lue trong 5 giir dau tian sau khi tiem \iiexiii vd nude mudi sinh ly. Ghi nhdn moi bieu hi?n vd thudng ciia cd thi nghiem. Tiep tyc theo ddi va ghi nhan boat dgng cua ca ciing nhu sd cd chdt hdng ngay cho dan ngay thii 21 sau khi ticm. Trong thdi gian ndy, cd dugc cho dn hdng ngay bdng thiic dn vien nhu da neu Iran va thay 30%i lugng nude trong be theo quy trinh chdm sdc ca tai Trung lam nghien ctiu gidng va dich benh thiiy sdn. Mgi cd chet thu dugc dau dugc kiem tra va phdn lap vi khuan tir nao va than. Ghi nhan cdc bian ddi vd thudng trong/tren ca the ca cd lidn quan den viec tiem vacxin.
2.4. Kiem djnh hl^u qua bao ve cua vacxin phdng benh do S. iniae tren cd chem 2.4.1. Gdy mien dich cho cd bdng vacxin vo hoat
Cd thi nghiem
Ca chem khdi lugng than trung binh 100 g diing trong thi nghiem nay dugc san xudt bdng con dudng sinh sdn nhan tao va uong nudi tai Trung tam nghien ciru gidng va dich benh thiiy sdn - Trudng Dai hgc Nha Trang. Ca khde manh, khdng cd tidn su cam nhiam vi khuan S. iniae va da thich nghi vdi ba nudi Ihi nghiem hi giai doan cd gidng cho dan khi bdt ddu su dung cho boat dgng nghian cuu.
Tiem vacxin hodc nirdc mudi sinh ly cho cd vd phdn biet cdc nhdm cd thi nghiem
Trudc khi tiam, ngung cho ca thi nghiem dn tdi thieu 24 gid. Khi tiam hdn dich, hmg dot ca thi nghiem (tiam vacxin vd ddi chiing) khodng 15 con / nhdm dugc bdt ra khdi ba luu giu bdng vgt, gdy me cd bdng dung dich Ethyleneglycol monophenylether 100 ppm roi tiem vacxin hodc
dung dich mudi sinh ly. Cd dugc tiem vacxin vao xoang byng (intraperitoneal injection - IP) vdi lidu lugng 0,3 ml/con, ca ddi chimg du^c tiem (IP) 0,3 ml dung dich NaCI 0.9%. Cd tiem vacxin vd cd ddi chirng dugc nudi dudng trong cdc be nudi rieng bigt vdi cung diau ki§n ba nu9i vd cha dd chdm sdc nhu nhau.
Dieu ki$n nudi du&ng cd thi nghiem:
Ca thi nghigm dugc nudi trong cdc 1*
composite dung tich 2m', chiia nude bien dp mdn 25-30%o, 6 nhigt dg phdng (27°C ± yQ).
Cho ca thi nghigm dn thirc dn vien (Seabass Feed - Uni-President Vigt Nam), mdi ngay 2 Ian, cho dn cham rdi bdng tay cho den khi cd thdi bdt mh\, ghi chep lugng thirc dn da sii dung sau mdi lan cho an.
2.4.2. Gdy nhiem thuc nghiem Mxdc ^nh hifu qud cua vacxin
Thi nghiem gay nhilm thuc nghiem de xac dinh hieu qua bdo ve ciia vacxin dugc hen hanh 3 tuan sau khi gay mian djch cho ca bdng each tiam 0,3 ml vi khudn S. iniae vao xoang byng, t6i thieu 30 con cd mdi nhdm (nhdm cd tiam vacxin va nhdm ca ddi chung). Mat do vi khudn trong lilu thir thach gdm 2 miic: 10' va 10* CFU/ml.
Thi nghiem dugc lap lai 3 ldn cimg thdi dilm - 3 Id cho mdi nhdm cd. Lieu thii thach sii dpg trong thi nghiem nay dugc xdc dinh bdng mgt kiam chiing bd sung dugc thuc hien trudc khi cdng cuong dgc nhdm dam bao ty lg chat ciia ca doi chiing dat tren 60%.
Ca dugc bdt tir cac ba nuoi bang vgt rdi gay ma trudc khi tiam vi khudn vdo xoang byng. Sau khi tiam vi khuan, cd dugc nudi riang trong cac ba composite Im' ddt trong phdng gdy nhiem thuc nghiem co diau khian nhiet dg dn dinh o 27"C bang may dieu hda khdng khi.
Hdng ngay cho ca dn thirc an vien, lugng thiic an theo nhu cau va hoat dgng bdt mdi cua ca, thay 50% lugng nude trong bd. Nude thay ra hi cic ba thi nghigm dugc gom ve ba xii ly, diet khuan bdng Chlorine SOppm trong 24 gid trudc khi bora vao he thdng xu ly chimg cua co sd thi nghiem.
KHOA HQC KY THUAT THLT Y TAP XX SO 3 - 2013
2.4.3. Quan sdt, theo doi cd thi nghiem, tdi phdn l^p vi khudn vd chdn ^dn benh
Thudng xuyen theo ddi cd thi nghi$m va ghi nhan cd chat trong cac ba 3 gid I lan. Ca thi nghiem dugc theo ddi lien tuc cho den khi khdng xuat hian cd chat trong mgi ba thi nghigm trong 7 ngay lian tuc.
Tat cd cd tiem vacxin Idn cd ddi chiing chet trong thdi gian theo doi cua thi nghiem gay nhiam thyc nghigm dau dugc giai phau, kiam tra vd phdn lap vi khuan tran mdi trudng nudi cdy chgn Igc (KF Streptococcus) dd xdc dinh
nguyen nhdn gdy chat.
2.4.4. Ddnh gid hlfu qud bdo vf cda vacxin Higu qud bao v? ciia vacxin dugc ddnh gid dya tran hg sd bdo ve tuong ddi (relative percentage protection - RPP) dya tren ty Ie chat cdng ddn ciia cdc nhdm ca dugc ham vacxin vd nhdm cd ddi chimg dugc tidm ciing mat dg vi khuan tai thdi diam kat thiic thi nghiem (7 ngdy ke tir ngay cudi ciing cd cd chat do vi khuan 5*. iniae gay ra trong cac ba thi nghigm). T^i thdi diem nay, gia tri RPP dugc tinh theo cdng thirc:
Ty If chit tich luy d* nhdm cd tiem vacxine
RPP = (I ) X 100 (%) Ty 1^ chit tich liiy 6* nhdm ca tiem nude muoi sinh ly
2.5. Xac dinh hdm Ivgng khang the ddc hieu trong mdu ca thi nghiem
2.5.1. Chuan bi cd thi nghiem
Ca thi nghiem su dyng cho viec kiem dinh bien dgng ham lugng khang the ddc hieu khdng S. iniae trong huyat thanh la mdt bd phan cua cd dugc tiem vacxin va cd ddi chiing trong thi nghiem xac dinh hieu qua bao ve ciia vacxin.
Sau khi tiam vacxin 28 ngay, 100 cd dd tiam vacxin dugc ham nhac lai lan 2.
2.5.2. Thu mau mdu cd thi nghiem Cd thi nghiem dugc thu hang tuan, ka tir ngay thii 7 sau khi hem vacxin, mdi ldn thu 10 con da dugc tiam vacxin va 10 con thudc nhdm ddi chiing tiam nude mudi sinh ly.
Gay me ca bang Ethyleneglycol monophenylether lOOppm rdi diing kim tidm I ml da Idy mau ca tii tinh mach dudi. Mdu cd dugc chuyen sang dng Eppendorf 2ml, bdo qudn qua dem d nhiet dd 4''C, tdch phdn huyet thanh tir mdu mdu va luu giu d nhidt do -20''C cho ddn khi sii dung da dinh lugng khanh thi dac hieu bdng ky thuat kilm dinh hdp phy mian djch lian kit enzyme gian tilp (Indirect Enzyme-linked Immunosorbent Assay- Indirect ELISA).
2.5.3. Kiem ^nh ELISA gidn tiep
Kiam dinh hdm lugng khang the ddc hieu khdng S iniae trong mau cd thi nghiem dugc tiam vacxin va mau ca nhdm ddi chiing dugc thuc hien bdng ky thuat ELISA gidn tiap theo phucmg phdp cua Crowther (1995) cd hieu chinh.
Cdc dia 96 gieng day bang dugc phii bdi hdn dich vi khudn S. iniae (40 yg/niL trong PBS; pH 7,3). Sau khi li qua dem d S^C, dia dugc nia 3 lan bdng dung dich PBS-Tween, khda cac vi tri lien kit khdng dac hieu bdng PBS-Tween chiia 3%
(w/v) skim milk trong 1 gid d nhiet do phdng.
Rira lai dia 3 Idn bdng dung djch PBS-Tween.
Huyet thanh cd thi nghiem dugc pha loang 50 lan, sau do pha loang dung dich ndy theo he thdng pha lodng he sd 2 (2-fold serial dilution) rdi cho huyet thanh da pha loang he thdng d cac miic khdc nhau nay vao cdc giang ciia dia 96 giang da chuan bj nhu tren. IJ dia d nhiet do phdng trong 60 phut, rira 3 Ian bang PBS- Tween rdi them dung dich khang tha don ddng cua chudt khang IgM ca chem (mouse anti-asian seabass IgM monoclonal antibody, AQUATIC Diagnoshcs Ltd.) da pha lodng d ndng do 1:40 trong PBS-Tween. U dia trong 120 phiit d nhiet do phdng, rira 3 lan bang PBS-Tween rdi them vdo cdc gidng dung dich khdng tha da ddng cua
KHOA HOC KY THUAT THU Y TAP XX S6 3 - 2013
da khang immunoglobulin cua chugt cd gdn kat peroxidase (peroxidase-conjugated goat polyclonal rabbit antimouse immunoglobulins HRD, Dako) dd pha lodng trong PBS-Tween (1:2000). U d nhigt dg phdng trong 60 phiit, nia 3 lan rdi ho^t hda peroxidase horseradish bdng cdch thdm 0-phenylenediamin (Sigma) vdo moi giang. Kai Ihuc phdn irng bdng cdch tham 50pl dung dich II^SOj 2N vdo gieng rdi do m§t dg quang (OD) d budc sdng 492nm bdng quang phd ke (Titertek multiskan MK II, Flow laboratories, Switzerland).
H I . K E T Q U A N G H I E N C U U 3.1. Tinh vd khudn ciia vacxin thu nghiem
Sau 48 gid nudi cay, khdng phdt hign thdy su xuat hien cdc khudn lac phdt trien tren cac mdi trudng nudi cdy NA, TSA, TCBS, vd KF Streptococcus cd bd sung 1,5% NaCI d nhiet do 28*'C. Kat qua ndy cho thdy, viec sir dyng formalin vdi ndng do 0,5%. dam bao vd hoat hda hodn todn cac te bao vi khudn S. iniae. Chd phdm thu nghiem nay cung khdng hi tap nhilm bdi Vibrio va cdc loai vi khuan khac.
3.2. Tinh an todn ciia vacxin thir nghiem do!
vol cd chem
Ca chem thi nghiem (khdi lugng trung binh I l,6±l,3g, chilu ddi than 8,3±0,4 cm), gdm 200 con dugc chia deu thanh 2 nhdm: nhdm dugc hdm 0,3ml vacxin vd nhdm dugc tidm 0,3ml dung dich nude mudi sinh ly. Sau khi tidm, mdi nhdm dugc nudi trong 1 hi composite dung tich 2m^ vd cham sdc theo quy trinh ky thudt neu h-an.
Trong 21 ngdy theo ddi ke tir khi tiem vacxin hoac nude mudi sinh ly cho ca thi nghigm, bien ddng cdc ydu td mdi trudng khdng cd su sai khdc Idn giira bl nudi ca tiem vacxin vd bl nudi ca ddi chirng. Nhiet do nude dao dgng tir 26 - 29''C; pH 7,6 - 7,8; hdm lugng oxy hda tan 5,0 - 5,4 rngOyi; do man dn dinh d 27%o. Ca thi nghiem bdt mdi tich cue vao ngay thii 2 sau khi tidm vacxin va nude mudi sinh ly.
Khdng phdt hign thdy cd chet d ca 2 nhdm ci tiem vacxin vd ddi chiing trong suot thdi gian theo ddi. Ty lg song dat 100%) d ca 2 nhomcS thi nghigm. Sau 21 ngay nudi, nhdm ca tiam 0,3 ml vacxin cd khdi lugng thdn trung binh 14,7
±2,3 g vd chilu ddi than trung binh d^t 8,7 ± 0,4 cm; cdc gia trj tuong ung d nhdm cd tiem 0,3 ml nude mudi sinh ly la 14,4 ±2,6 g vd 8,5
± 0,5 cm.
Kat qua thyc nghigm cho thay vi^c tiem vacxin vd hoat thu nghiem vdo xoang byng cua ca chgm thi nghigm khong gay chat ca vd kh6ng dnh hudng din sinh trudng cua cd thi nghifm so vdi cd ddi chung. Tuy chidu dai vd khdi lugng than trung binh ciia nhdm ca dugc hem vacxine thdi diem ket thiic thi nghiem cao hem nhdm ca doi chung dugc ham nude mudi smh ly nhung phdn tich thdng ke cho thay su sai khdc ve kich thudc giiia 2 nhdm cd thi nghiem khdng co y nghTa vd mdt thdng ke (p<0,05). Mat khdc, khi md 30 ca the d mdi nhdm d^e quan sdt xoang hung cd tai vi tri tiam vacxin, khdng quan ii\
thdy nhiing bien ddi vd thudng hodc su hinh thanh cac khoi u tren thanh byng hodc phiic mac. Vi vdy, cd thd nhan dinh rang vacxin thir nghiem la an toan ddi vdi ca chem vd cd the sii dyng da gdy mien dich va kiam dinh hi^u qua bao ve cua cha phdm thii nghiem ndy trong vifc ngan ngira Streptococcosis tren ca chem.
3.3. Hi^u qua bao vf cua vacxin thir nghifm doi vdi cd chSm
Mgt ngdy sau khi cdng cudng ddc cdc nhom ca thi nghigm bdng cdch tiem 0,3 ml hon dichvi khudn S. iniae chung VN09I2IIR d cdc m?tdo 10' va IO'' CFU/ml vao xoang byng cua ca, dci;
Id cd ddi chimg cd hien tugng ngirag bit mfii.
Cd chat vdi ddu hi$u dac trung cua b?nh do S.
iniae gay ra (Idi mdt, xudt huylt d gdc vay nguc vd vay duoi) bat dau xuat hign d cdc be nufii cd ddi chiing vao thdi diam 36 gid sau khi gSy nhilm vm mat dg vi khuanlO^ CFU/ml (Hbili 1). Sd luofng ca chit tdng vgt d cac bl ndy iSng nhanh trong khoang thdi gian hi 48-72 gid sau
KHOA HQC KY THU^T THU Y TAP XX S6 3 - 2013
khi tiem vi khuan, va dat gid tri ty lg chit cgng ddn d thdi dilm kit thuc thi nghigm Id 83,33%.
C) cdc ba nudi ca ddi chung dugc cdng cudng dpc vdi mat dg vi khudn 10' CFU/ml ciing cd hi?n tugng tuong tu, tuy nhien, ca chi bdt dau chit til 2 ngay sau khi cong cudng ddc vd ty le chit tich luy tdi da Id 60%). Ca chit vdi dau higu
dac trung cua Streptococcosis cung dugc phdt hign trong cac nhdm ca dugc ham vacxin trong khodng thai gian hi 36 - 72 gid sau khi cdng cudng ddc. Tuy nhian, ty lg chet cgng ddn trong sudt thdi gian theo doi thi nghiem chi dat 20%i d nhdm tiem hdn djch 10" CFU/ml va 6,66% d nhdm tiem hdn dich 10' CFU/ml.
Hinh 1. Ty 1$ ch^t tich luy d cdc nhdm cd thi nghi$m trong thdi gian theo doi sau khi cdng cudng dQC bkng vi khuin S. Iniae chung VN091211R (V7, V8: cA ti§m vacxin, cdng cudng d0c vdi hdn dich vi khudn mSt dd IO' CFU/ml ho$c 10^ CFU/ml; C7, C8: cA doi chiing. cdng
cudng ddc vdi hdn dich vi khuin mSt dd 10' hoScIO^ CFU/ml)
12 24 36 48 72 84 96 108 120 132 144
Thdi gian sau khi cdng cudng ddc (gid) He sd bdo ve tucmg ddi d cac nhdm ca thi
nghiem tuong iing vdi cudng do cdng cudng dgc la 89% (10' CFU/ml) va 76% (10« CFU/ml).
Cac gia tri ndy deu cao hon mire hieu qud quy udc (RPI^ 60%) khi kilm djnh hieu qua bdo ve cua vacxin d ca (Amend, 1981), va thda man cac diau kian ve ddnh gid hieu qud vacxin diing cho ca ciia IJy hgi Chdu Au (Commission of the European Communities, 1998) do dd, vacxin thir nghi?m nay cd tac dung bao ve ca chem chdng lai b?nh do vi khuan S. iniae gay ra. Tuy nhian, so vdi mdt sd loai vacxin phdng benh cho cd dang luu hanh d nude ngoai vdi he sd bao ve dat tran 90% (Elda et al., 1997), hieu qua chi phdm vacxin thir nghiem cua nghian ciiu nay van cdn thap. Be nang cao hieu qua ciia viec dp dyng cua
che phdm ndy, cdn xem xet viec phdi hgp chat bd trg (adjuvant) vd/hoac sir dung cdc chat midn djch khdng ddc hieu, dac biet la p-glucan, da ndng cao hieu qua boat dgng cua he mien djch khdng dac hieu trudc khi gay mien dich cho ca.
Ngoai ra, thdi gian bao hg ciia lieu phap vacxin nay cung cdn duoc tiap tuc kiam nghiem nham dam bao cha phdm vacxin ndy cd tha bao ve cho ca trong sudt vu nudi.
3.4. Bien dong hdm lugng khang the dac hi^u trong mdu cd thi nghiem
Sau khi tidm vacxin 7 ngay, ham lugng kliang the dac hieu khang vi khuan S. iniae trong huyat thanh ciia ca tdng va sai khac rd ret so vdi nhdm cd ddi chiing (Hinh 2).
KHOA HQC KY THUAT THU Y TAP XX S 6 3 - 2013
i I
Hinh 2. Bien dgng ham lupng khdng the d0c hi$u khdng vi khuan S. iniae trong huyet thanh cua Cti chem dupe tiem vacxin thi^ nghi$m so vdi cd doi chufng (OD^^^J
Hdm lugng khang the irong huyel thanh dai cue dai vao ldn ihu nidu tiep theo (ngdy 14 sau khi ga\ mien djch) va cd xu hudng giam dan trong kin thu mdu thii 3 (ngay 21 sau khi lidm vacxin). Sau khi duoc tiem vacxin nhdc lai vdo ngay 28, ham lugng khang the dac hieu trong mdu ca lang nhanh vd dat cue dai d lan thu mau thir 6 (ngay thir 14 sau khi tiem nhdc lai) vdi ham lugng khang thd cao gap 2,5 lan so vdi ham luOTig khdng the cue dai trong dap irng mian dich so cap.
Bien dgng ham lugng khang tha ddc hiau trong cac nhdm ca thi nghiem cho thay rang, hieu qud bao ve cd chem dugc tiem vacxin tran da> Id he qud dap iing mien dieh dich tha ddc hieu cua ca thi nghiem ddi vdi khang nguyen dac trung cua chung vi khudn gay benh.
IV. KET LUAN
Che pham vacxm vd hoat san xiifil hr chung VI khuan S luiac phdn lap tir cd benh tai Khanh Hoa id vo khuan, an toan, vd cd tac dung bao ve dac hieu cho ca ihi nghiem. ngdn ngira benh do VI khuan ndy gay ra tren ed chem nudi vdi he sd bao ve luong ddi cao. Tuy nhien, de cd the phdt trien che pham nay thanh san pham thuong mai phiic vu phat tnen ben viing nghe nuoi ca chem tai Viet Nam, can liep luc nghien ciiu de nang cao hieu qua bao ve vd xac dinh duoc thdi gian bao hd ciia vacxin.
TAI LIEU THAM KHAO
I, Amend D F (1981) Potency testing of fish vacc Developments in BiDlogieal Siandardization 49, -454
3 Commission of the European Communities (1?
Specific requirements for the production and coin live and inactivated vaccines intended forfi^ In:
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Union. Guidelines for ihe testing of veteri medicinal products Volume VII, pp 245-250.
4 Eldar A.. Horoviiz A & Bercovier H (1' Development and efficacy of a vaccine agi Stivpiococcus tniae infection in farmed rainbowjj li'termun' Immunology and ImmunopatholoN
J-.^-IS] 1 5 Kusuda R . Kjwai K., Toyoshlma T. and Komals
l')76. A new pathogenic baclenum belonging to genus Sirepiacocciis. isolated from an epizootii cultured yellowtail.yp/j Soc Scj Fi.v/i, 42T345-13J 6 Ph Eur monograph: 1581: Vibriosis vac
(inaclivaled) forsalmonids.
7, SiolVregen DA. Backman SC. Perham RE, Bowser BabishJGl 1996) inilial disease report of5rre/)roco iniiif infection in hybrid striped (Sunshine) and the successfiil therapeutic intervention tluroquinolone antibacterial enrofloxacin. J. W Aqiici. ,9(.[ 2^ 4:0-4i4
8. Weinstem. M. R. R. (1997) "Invasive infeclions due to a fish pathogen. Streptococcus miae S. iniae Sfiidy Group •" The New England journal of medicine 337(9):
Nhan ngay: 5.2.2013 Phdn bien ngay: 15.2.2013
