KHOA HOC KY THUAT THU Y TAP XIX S6 7-2012
KHA NANG THiCH iTNG COA M Q T SO CHUNG VIRUT BAU D£ (PHAN LAP
fliTdfc Tir T £ BAG TINH HUAN n£) T R £ N CAC LUAI li BAU sd CAP
THAN D £ , THAN ciru, TINH HQAN CUU VA l€ BAU VERU
DS t an Khien', Phgm Hung', Le Thanh Hoa', Nguyin Viit Khdng' T 6 M TAT
TCr 7 chiing vimt dgu dd dd qua phdn ]0p trdn tl bdo tinh hodn dd, tilp tyc dupe kilm tra khd ndng thich img ciia chiing tren cdc logi tl bdo S(T cap tinh hodn cuu (LT), te bdo th§n dd (GK), thgn cuu (LK) vd te bdo ddng thuIn vero Ket qud kidm tra cho thiy cdc chiing vimt ndy thich ung tdt tren cdc logi tl bdo ndi tren, gay bdnh ly tc bdo (CPE) d ngdy thu 4 vd thu 5 sau khi gdy nhilm, d lin tilp tmyen thu 6 vd thu 7. Chi soTCIDSOdgtcaonhltkhi cdc chiing vimt ndy thich ung trdn tl bdo vero, kl den te bdo LT. tc bao LK vd GK.
Tir khoa: Vimt dgu dd, Tl bdo tinh hodn ciru, Tc bdo thgn dd, T I bdo than cim , Te bdo vero, Kha ndng thich img.
Adaptability of the goat pox virus (isolated from tlie goat testicular cells) on the goat Icidney, lamb icidney, lamb testicular primary cells and vero cell line
Do Van Khien, Pham Hung, Le Thanh Hoa and Nguyen Viet Khong
SUIVIMARY
7 goat pox virus strains isolated from goat testicular cells were tested their adaptability on the lamb testicular primary cells (LT), goat kidney cells (GK), lamb kidney cells (LK) and vero cell line. The testing results showed that all of 7 virus strains adapted well to the above cell culture types and caused cytopathic effect (CPE) at day 4'" and 5"* after inoculation in the passage 6""
and 7'^ The TCID50 index reached the highest value when these virus strains adapted on vero cells, following by LT, GK and LK cells.
/Cey words; Goat pox virus, Goat kidney cell, Lamb kidney cell, Lamb testicular cell, Vero cell, Adaptability.
I. D A T V A N D E
Bdnh dgu de, cim Id bdnh tmydn nhiem cap tinh cho dd, cim, d$c tnmg bdi s^r Idy lan nhanh vd ty Id chit tdi 50%, cd tmdng hpp ty 1? chit trong ddn gia sue non len din 100%. Bdnh dupe Td chiic dich te thl gidi (OIE) xlp vao bdng A danh muc cdc bdnh nguy hiim. Cdn bdnh do mpt loai DNA vimt thupc gidng Capripoxvirus, hp Poxviridae gay ra. Bdnh xay ra d nhilu noi
trdn the gidi nhu Bdc Phi, Tmng Ddng, An Do, Nga, Mdng Cd, Tmng Qudc va Vipt Nam, gay nhidu thidt hgi vd kinh tl cho nganh chdn nudi dd, cim. O Vidt Nam bdnh xuat hipn d gt vao 2005 tgi mpt so tinh phia Bdc nhu Cao Bdng, Lgng Son, Bdc Giang, Ha Tay. Sau dd bdnh nhanh chdng lan ra cdc tinh ed chdn nudi de thupc mien Tmng vd mien Nam gay dnh huong nghidm trpng den chdn nudi dd d nude ta. De c6
' Phan vi?n thii y Mien Tmng
^ Vi^n Cdng ngh? sinh hpc ' Vidn thii y
18
KHOA HOC KY THUAT T H U Y TAP XIX S6 7 - 2012
nhihig diJ lipu nghidn curu vl ddc tinh sinh hpc cua vimt dgu de d Vidt Nam, trong nghien ciiu nay chiing tdi tien hdnh ddnh gid khd ndng thich ling cua mpt sd chiing vimt cudng dpc d§u dd frdn mpt so logi t l bdo so cap tinh hodn cdu.
than de, thgn cim vd ddng tl bdo vero dl tdm tiln de cho cdc nghidn cixu tiep theo vd vimt daude d Vipt Nam.
II. NGUYEN Lifu VA PHUONG PHAP
2.1. Nguyen lifu
2.1.1. Te bdo, moi tru&ng nuoi cay - Cdc logi te bdo so d p tinh hoan cim, than de, thgn cim vd te bao ddng thuIn vero do Phdn vien thii y midn Tmng cung d p .
- Mdi trudng nudi cdy te bdo EMEM bd sung 5% huydt thanh bdo thai be.
2.1.2. Kit chiet tdch DNA vd primer - Sir d\mg bp kit QIAamp DNA minikit cua hang QL\GEN dimg de chiet tach mdu ADN tdng sd tir mdu dich tl bao nudi cay vimt dau de.
- Cdp mdi gidm dinh vimt ddu de trong phan dng PCR gdm;
Mdi xudi PIF: 5"- TTT CCT GAT TTT TCT TAC TAT -3'
Mdi nguoc P2R: 5'- AAA TTA TAT AGC TAA ATA AC -3'
2.1.3. Nguon virut d$u de
- Gdm 7 chung vimt dgu de dd qua phdn lap tren td bdo tinh hodn dd so cap.
2.2. Phinmg phdp nghien cihi 2.2.1. Gdy nhiSm virut trSn te bdo
Quy trinh gay nhiem vimt d$u de tir bdnh phim hodc vimt da qua phdn lap dupe thyc hidn tren cdc logi t l bao tinh hodn de, cim so cdp; te bdo thdn dd, cihi so d p va tl bao vero tai phdng thi nghidm vimt, Phdn vien thu y miln Tmng.
2.2.2. Phmmgphdp thich ung cda virut O^u dS trin moi tnrdng t& bdo
- V§t lidu tiep ddi Id mau vimt phan lap duong tinh trdn te bdo tinh hodn dd,
- Cac logi te bdo sir dyng trong tilp ddi gdm: tl bdo tinh hodn ciru, tl bdo than de, cim so d p vd te bdo vero.
- Thi nghidm tilp ddi dupe tidn hdnh trdn chai T25 (didn tich ddy 25cm^) theo quy trinh gay nhidm ndu trdn day.
- Sd lin tilp ddi Id 7 Idn. Huyen djch td bdo nhiem vimt dupe thu vd chuan dp de xdc djnh hieu gid vimt (TCID50).
2.1.3. Phuang phdp xdc djnh hipu gid virut (chi so TCID50)
Dupe tidn hdnh theo phuong phap thudng quy phdng thi nghidm. Kit qua TCID50 dupe tinh todn theo cdng thiic Reed-Muench.
2.1.4. Phuang phdp gidm djnh virut dau de - Tach chiet ADN tong sd tir mlu vimt gay nhidm tren te bao theo hudng din cua nha san xuat kit QIAamp DNA minikit cita hang QIAGEN.
- Chay PCR sir d\mg cap mdi PI va P2 theo thilt kl cua Ireland va Binepal (1998) cho san phim PCR cd dp ddi Id 172bp (san phim PCR da dupe xac dmh chinh xdc sau kdt qud gidi trinh tvr gen P32 ciia vimt dgu de).
I I I . K E T Q U A VA T H A O LUAN 3.L Kha ndng thfch iing cua virut dau de tren t l bdo tinh hodn cim so* d p
7 chiing vimt cudng ddc dau de phan lap lln diu hjr thirc dia h-dn tl bdo GT (Vn07Ntl, Vn07Nt3, Vn07Nt4, Vn08Nt6, Vn08Kh9, VnOSKhlO, Vn08Py7) dupe tilp ddi tten mdi frudng tl bdo tinh hoan cuu (LT) so cap . Chung tdi sir dyng chai tl bao LT (didn tich ddy 25 cm^) dgt dp phii tren 80% tl bao; huydn dich vimt tgi lilu 200 (il cho 1 chai te bao. Kit qua ddnh gid khd ndng thich img ciia vimt trdn tl bdo tinh hodn dvra vdo thdi gian sdm nhdt xudt hidn CPE dupe trinh bdy d bdng 1 va hinh dnh bdnh ly t l bao dupe md td d hinh I.
19
KHOA HOC K? THUAT THO V TAP XIX S67-2012
Bing 1. Qui trinh thich ling virut atu M Uin \i Mo so cip LT S6 ddi gfly nhiim
1 2 3 4 5 6 7 K^t q u i g l i m dinh PCR
Ngjiy xud hl«n CPE 9 8 7 6 5 4 4 (+)
Chai li b i o LT c6 CPE 7/7 6/7 6/7 7/7 7/7 7/7 7/7 7/7
C/j; chii: Lieu gdy nhiem 200 fil dich nuoi te bdo chiia virut cho chai 25 cm' Kdt qud trinh bdy d bdng I cho thdy:
- Sau mdi lan cay tmyen, thdi gian xudt hidn bdnh ly t l bdo ngdn hon: Ddi vdi te bdo tmh hodn cim, sau 4 ldn cay tmydn, ca 7 chung vimt cd thdi gian xuat hipn bdnh ly td bdo dupe nit ngan tu ngdy thu 9 xudng ngay thii 6. Td Idn cdy tmydn thu 5 xudt hidn bdnh ly td bdo d ngdy thii 5; sau lln d y tmydn thir 6 vd 7, xuat hidn bdnh ly d ngdy thu 4 sau gdy nhiem. Kit qua nay chiing td 7 chung vimt dgu dd phdn lap cd khd ndng thich img cao vdi tl bdo tinh hodn cim.
Trong qud frinh quan sdt bpnh ly te bdo dcac chai gay nhiem tu ddi thu 1 din thii 4, chiing tdi nhgn thiy 2 chiing GpvPy7 vd GpvKhlO thudng xudt hidn bdnh ly td bdo chgm hon so vdi cdc chiing khdc. Tuy nhien d cdc ddi tir thii 5 den thti 7, cac chiing vimt ndy gay bdnh ly td bao dn dinh.
- Bdnh ly td bdo d te bdo LT dupe minh hpa d hinh I.
Hinh 1. B\in 66\ b#nh ly d Xi bio LT
Hinh anh le bao LT hinh thuong (A) va te bao LT vai CPE, ngay thir 4 sau gay nhiim a lin thii 7 nip dai lien tiip virut dqu de (B): hinh anh difn di san pham PCR phdt hien DNA vimt dgu de a cic chai li bao sau 7 lan tiip dai: Giing M: thang DNA chudn: giing I. 2, 3. 4, 5, 6, 7: san phdm PCR twang img vai 7 chung virut dgu de Vn07Ntl, Vn07Nt3, Vn07Nt4, Vn08Nt6, Vn08Kh9. VnOSKhlO, Vn08Py7 dd qua 7 ldn tiip dai tren ti bao LT.
KHOA HOC KY THUAT T H U Y TAP XIX S(5 7 - 2012
0 ngay thii- 8 sau gdy nhiem, phdn ldn cdc chai td bdo cd CPE dgt hi (-H-) din (+++). Thdi dilm ndy chung tdi thu hogch tit cd cdc chai tl bdo cd CPE bdo qudn d -30''C. Ddi vdi djch nudi cay te bdo sau lln d y tmyen thu 7, chdng tdi chiSt tach ADN vd kidm tra sy cd m$t cua vimt trong huyIn dich td bdo nhidm vimt bdng phdn img PCR, sir dyng c$p mdi PIR-PIF d$c hidu cho nhan gene p32.
- Kdt qua didn di sdn phim PCR d hinh I cho biit duong tinh PCR d tit ca cdc mau vimt sau 7 Idn tiep ddi, chung Id bdnh ly te bdo d moi lln tidp ddi (cd tmde dd) ddu Id do ddc tinh gay bdnh ly te bdo ciia cdc chiing vimt dgu de.
- Nudi d y vimt dgu cim, dgu de tren tl bdo tinh hodn sdm dupe thilt lap tu ndm 1957 d mpt logt cdc phdng thi nghidm nhu d Italia, d Phdp, d Anh ndm 1958. Hluhltcdc nghien ciru phan Igp ddu su dyng te bdo tinh hodn cim. 0dc tinh cua vimt ddu dd thich nghi frdn tl bdo tinh hodn de va tmh hoan cim dd dupe sdm xdc nhgn bdi Hess va cs (1963). Tiep ddi vimt dau de phdn lap frdn tl bdo tgi thuc dia nhdm dn dinh s\r thich nghi ciia vimt frdn t l bao nudi, gift gidng vimt nghidn cdu cdc chiing nhupc dpc, nghien cuu tdng sinh lupng vimt nhdm san xuat vacxin (Ramisse va es, 1978; Ramesh 1980; Bhanuprakash vd cs, 2003; Zhou vdcs, 2004).
Kit qud cho thiy cd 7 chung vimt d§u de deu cd cung ddc tinh nhanh chdng thich ung vd dn dinh frdn tl bdo so d p tinh hodn cim. Sau 7 lln tilp ddi lien tilp, ca 7 chung ddu nhan Idn nhanh chdng tren tl bdo, sau 5 ngdy bdt diu xult hidn bdnh ly tl hdo vd sau 8 ngdy dgt muc (-t-H-). Th\rc hidn 2 lln ldp Igi ddu cho kit qud tuong ducmg.
Sau 7 lln tilp ddi lien tilp, chiing tdi dd thu dupe cdc chiing vimt thich img tit vd dn djnh , cd kha ndng bdo tin su dyng nhu nguon gene vimt phyc vy cdc nghidn cuu kl tilp.
3.2. Kha ndng thich irng cua virut dau de tren t l bdo th§n sff d p
Vipc nghien cuu khd ndng phdt trien vimt d cdc logi td bao khde vdi te bdo tinh hodn cd y nghia tim ngudn thay thd cho tl bdo so d p tinh hoan cim vd tinh hodn de.
Td bdo than de vd than ciru so cap dupe chudn bi tu dd vd cim 3-4 thdng tuoi. Td td bdo tidn phdt chiing tdi da cay chuydn vd dn dinh sau 8 ddi fren moi tmdng EMEM vdi 5% huylt thanh bao thai bd. Ngudn vimt gdy nhidm lln diu Id 7 chiing vimt da phdn ldp frdn tl bao tinh hodn de (200 \i\ gdy nhidm cho 1 chai te bdo than so cap T25); sau dd sir dyng 200 n\ cho mdi Idn gdy nhidm ke tiep. Ket qud xac dinh CPE vd theo doi thdi gian xuat hipn CPE sdm nhdt dupe trinh bdy d hinh 2 va bang 2.
Hinh 2. B^nh ly 6 tS' bao than d6 vk th$n cCfu
Ti bao than de: (A) binh thudng. (B) co CPE; le bdo thgn ciru: (C) binh thudng, (D) co CPE
KHOA HOC KY THUAT THU Y TAP XIX S6 7 - 2012_
Qua hinh 2 cho thiy:
- So vdi tl bdo th$n binh thudng khdng gdy nhilm vimt. cdc lc bdo th$n de vd th$n cCru gdy nhidm vimt d§u dc cd biln d6i rO rdt, cdc tc bdo CO cym vd d$m mdu. D$c diem hinh thdnh thl viii dgm mdu DNA Id m^t trong nh&ng djc dilm
nudi d y vimt d$u capripox frdn td bdo, - Si; xult hidn bdnh ly te bdo sdm hay muOn, mdc CPE (+) den {+++) tdy thupc vdo chiing vimt vd thdi gian nudi d y . Thudng sau 72 gi6 tir khi xult hi§n CPE, biln doi bdnh ly phii to4n bO ldp t l bdo.
Bdng 2. Qud trlnh thfch tirng virut d$u dd tr6n t6 bdo th^n d6 so dp Siddl
gSy nhiim 1 2 3 4 5 6 7 K«t q u i g i i m dmh PCR
Ngdy xuit hl$n CPE
8 8 7 6 5 5 5 (+)
Chtllibto GK CIS CPE
6/7 5/7 6/7 6/7 6/7 7/7 7/7 7/7
Chai le bio LK c6 CPE
5/7 6/7 6/7 7/7 6/7 7/7 7/7 7/7
Ghi chu: Lieu gdy nhiem 200^1 dich nuoi te bdo chira virut cho chai 25cm' Ket qua bang 2 cho thdy:
- Cd 7 chiing vimt gay nhiem deu phdt inen tdt frdn te bao than dd, than cim vd gdy bdnh ly tl bao sdm nhat vao ngdy thii 8 d ldn dau tidn va lln thu 2 sau khi gay nhilm.
- Tir lln gay nhiem l§p Igi thu 3, cd 6/7 chiing vimt cd bdnh ly tl bdo d cd 2 logi tl bdo GK vd LK (chiing PY7 cd CPE ch§m hon). Tuong t\t d cdc ddi gdy nhiem tidp theo, ddi 4 vd 5 ciing cd 6/7 chung ddng logt xult hidn CPE tuong img vdo cdc ngdy thir 6 vd 5 sau gay nhilm (vimt PY7 vdn Id chiing gdy CPE chdm hem so vdi cdc chiing vimt khdc).
- Ket qua theo ddi tir ddi thii 6 din thii 7, tdt ca 7 chiing vimt gdy nhiem ddu cd CPE trdn 2 logi tl bao GK vd LK d ngdy thu 5. Ket qud kiem fra sy cd mdt cua vimt bdng k j thugt PCR phdt hien ADN vimt ddu d cdc mau djch nudi cua hai loai te bdo ndy d lln tilp ddi thu 7 cho biet: Cd 7 chung diu cho sdn phdm PCR cd dO dai dogn gen Id 172bp, duong tinh vdi vimt dgu dd (khdng trinh bdy kit qud).
Td bdo than cim thudng dupe su dpg frong d y tmyen vimt ddu cim (Pandey va cs, 1969, Bhanuprakash vd cs, 2003). Nhin chung vimt ddu cihi phdt trien dn djnb frdn tl bdo LK vd GK, cho CPE ddc hidu ciia cdc capripoxvirus (Adiakha vd cs, 1971) Tuong t\r, vimt dgu dfi cung cho kit qud nudi d y dn djnh tren te bdo thgn dd vd thgn cim (Joshi vd cs, 1994). 6 Vi?t Nam, Trin Thj Lien va cs, (2008) ciing dd ^p dyng thdnh cdng nudi d y vimt dgu dd fren te bdo GK va LK.
- Vimt capripox cdn cd khd ndng thich ung fren cdc t l bdo dj lodi nhu tren td bdo thdn bd so sinh, tuy nhidn hi§u gid thudng thdp hon.
Ngupc Igi, nhihig tl bdo cimg loai, ngay khi dd dupe ddng hda thudng ddm bdo s\r thich ung vd nhan len d hi?u gid cao (Babiuk va cs, 2007).
3.3. Khd ndng thfch irng cua virut dau d§
trdn ddng tl bdo vero
Td bdo vero dupe biit Id t l bdo cdm thg ciia vimt dgu de, ddi khi cd the su dyng frong lln phdn Idp diu tidn nhu ddi vdi d djch tgi 22
KHOA HOC KY THUAT T H U Y TAP XIX S<5 7 - 2012
Makhdoom, bang Uttar, An Dp ndm 2008, hodc o djch ddu dd 2005 d Vidt Nam. Trong nghidn cdu nay, chiing tdi khdng phdn ldp dupe virut frdn te bdo vero d lln phan l§p diu tidn. Tuy nhidn, de xdc nhdn d§c tinh vimt dgu de phdn Igp tgi Vipt Nam, ddng thdi ed ngu6n tl bdo ddng de cay chuyen vimt trong cdng tdc luu gift ngudn vimt, san xuat khdng nguydn vimt sau ndy, chung tdi thu tinh thfch ung ciia 7 chiing vimt dgu dd thyc dia, sau khi dd phdn I§p lln diu fren tl bdo tinh hoan de.
Phuong phdp gdy nhiem vimt cho te bdo vero (chai T25) trong tilp ddi lien tilp dupe tiln hdnh nhu dd md td tai phan vgt lidu vd phuong phdp vd tuang ty nhu doi vdi tl bdo tinh hodn cim, tl bdo than de vd than cim trinh bdy trdn ddy. Vat lidu gdy nhiem cho moi chai te bdo T25 Id 200 ^il huydn dich vimt tir huyen djch phan Igp vimt tren tl bdo tmh hodn de (lln tiep ddi I) vd td lln d y tmyen tmde (cac lln tilp ddi ke tilp). Kit qud quan sdt bpnh ly te bdo dupe minh hpa d hinh 3 vd theo ddi thd!
gian xult hidn CPE dupe trinh bay d bdng 3,
Hinh 3. Hinh dnh b^nh ly d te bao virut d$u d£ tren t§' bao vero (A) te bdo trudc khi gdy nhiem virut ddu de, (B) te bdo co CPE
Bdng 3. Qud trinh thich iJng ci^a virut dgu de tren t^ bao vero S6 ddi giy nhiSm
1 2 3 4 5 6 7 K ^ t q u i g i i m dmh PCR
Ngiy xu^t hl$n CPE 8 7 7 6 6 5 5 (+)
Chai tSbio vero c6 CPE 5/7 5/7 6/7 6/7 6/7 7/7 7/7 7/7 Ghi chu: Lieu gdy nhiem 200fxl dfch nuoi te bdo chira virut cho chai 25cm' Kdt qud hinh 3 vd bdng 3 cho thdy:
- Khdc vdi d Ian phdn Igp dau tidn, khdng cd SI; khdc biet giiia hinh thdi td bdo d chai te bdo vero ddi chdng am (khdng gdy nhilm bdnh phim dau dd) vd d cdc chai cd gdy nhidm huydn
dich bdnh phdm dgu de, theo doi ddn 13 ngdy sau gay nhidm.
- Sau khi gay nhidm 8 ngdy bdng vimt phdn ldp trdn td bdo tinh hodn de, bit ddu xudt hi?n cdc ddm te bdo co cym, ria tl bdo khdng r5, 23
KHOA HOC KY THUAT T H U Y TAP XIX S6 7 - 2012
d§m mdu [ddnh gid CPE (+)]; din ngdy thir 9, so CTI tdng din, tgi nhOng cym d§m mdu, khdng thiy ria tl bdo, chi thiy nhdn vd cdc the viii thim mdu. d viing sdng mdu vln cdn nh&ng tc bdo cdn rd hinh thdi [ddnh gid CPE (++)];
den ngay thir 10 sau gdy nhilm, s6 CPE tdng den (+++) phii phan ldn thdm tl bdo.
- Ket qud theo ddi thdi gian xult hipn CPE d cac chai tc bdo vero tilp ddi virut d§u dd cho thiy:
0 lln d y tmyen thu nhat. cd 5/7 chimg vimt phdn Igp gay bdnh ly te bdo d ngdy thu 8, hai mlu cdn lai. bdnh ly tl bdo xuat hidn mu^n han vdo ngdy thu 9 (mlu GpvPy? vd (i|nKhlO), nhu \ay, cd 7 chimg vimt dgu dd diu phdt tridn trdn ddng te bdo vero.
C) ldn cay tmyen thii hai vd ba, tgi 7 ngdy sau gay nhidm, cd 5/7 sau dd Id 6/7 mlu xult hien CPE ddng logt vdo ngdy thu 7; cdc mlu xult hidn CPE chdm hon van Id GpvPy7 vd GpvKhlO.
O lan cdy tmydn thu tu va thu ndm, tgi 6 ngay sau gay nhiem, cd 6/7 mdu xudt hipn CPE ddng logt vdo ngay thii 6; mau xudt hipn CPE chdm hon la GpvPy7.
O ldn cay tmydn thir sdu vd thu bay, todn bd 7 mau vimt diu gdy CPE xult hidn vdo ngdy thii 5 sau gdy nhiem.
- Kit qud kilm tra ADN vimt dgu d^ bdng phuong phdp PCR sir dyng cdp mdi PIF-PIR ddi vdi cdc mlu d y tmydn vimt ddi thu 7 diu xdc nhan sy cd mdt cua vimt dgu dd.
- Kdt qud cay tmyin sau 7 lan vimt dgu de fren td bdo vero vdi bpnh biln te bdo dien hinh, PCR xdc nhdn si; cd mdt ciia vimt sau ddi d y tmydn thii 7, cho phdp kit ludn, chung tdi dd thu dupe vimt dgu dd thich iing frdn tl bdo
vero. NhOng chung vimt thich iing tren tl Uo vero dupe tilp ddi din lln thii 9 (khdng dan si li^u), thdi gian xult hipn CPE on djnh d ngay thu 5 sau gdy nhilm vd hidn dupe bdo qudnd -86"C.
- Cdc nghidn cuu trdn thl gidi vdi myc dich sdn xult vacxin dd thdnh cdng su dyng td bdo vero dl thich ting vimt d^u dd vd dgu ciru (Singh vd Rai, 1991a, Ved Prakash vd cs, 1994); thich dng vimt d0u cim (Yogisharadhya vd cs, 2011);
thich irng vimt ddu dd (Hosamani vd cs, 2004);
Khdc vdi mOt s6 nh^n djnh vl hi§u gid virut ircn tl bdo vero thudng thdp hon frdn tl bao tinh hodn cuu gan ddy, vimt dgu dd tiep ddi trdn tl bdo vero cd hidu gid on djnh d mirc cao (xem kit qud TCID50 d phin sau), phdn dnh mpt d$c tinh nudi cay cd the khdc vdi cdc chiing tren the gidi vd la ling ciia vien cho chung vacxin trong nhihig nghien ciiu kd tiep.
3.3. Kit qud xdc dinh hi$u gid virut (TCID50) ciia virut phdn ldp
Trong sd 7 chiing vimt dgu de dd phdn Idp vd tilp ddi fren cac ddng tl bdo trdn, chiing toi nhgn xdt cd 2 nhdm kieu gdy CPE: nhdm gay CPE nhanh gom 5 chung (Vn07Ntl, Vn07Nt3, Vn07Nt4, Vn08Nt6 vd Vn08Kh9) vd nhdm gdy chgm cd 2 chiing (GpvPy7 vd GpvKhlO).
Dd xdc djnh hipu gid cua cac chiing vimt sau tilp ddi, chung tdi sii dyng 3 chung d nhdm 1 (Vn07Ntl, Vn07Nt4, Vn08Kh9) vd cd 2 chiing d nhdm 2. Te bdo chuan dp Id tl bdo ma chiing vimt da thich nghi gdm GT, LT, GK, LK va vero, vimt chudn dp la huyen djch cay tmydn ddi thu 7. Chuan dp vimt dupe tien hdnh nhu md ta d phin v§t lipu va phuong phdp, su dyng 9 dp pha lodng vimt (td 10' din 10'), thyc hi?n fren dia nudi cay te bao 96 gidng khi td bdo phii ddy 80%. Kit qua chudn dp hipu gia virut (TCID50) dupe hinh bay d bang 4.
KHOA HQC KY THUAT T H O Y TAP XIX S6 7-2012
Bing 4. Hl$u gii virut (TCID50) cOa cic chfing virut d|u 0§ tr«n ti bio TT
1 2 3 4 5
Chiing virut Vn07Ntl Vn07Nt4 VnOBKhg Vn08Kh10 Vn0ePy7
Chl sd TCID50 (loglO) trin cic loai It bio GT
10'^
1 0 "
1 0 "
1 0 "
I O "
LT 1 0 "
1 0 "
1 0 "
1 0 "
1 0 "
GK 1 0 "
1 0 "
10<' 1 0 "
1 0 "
LK 1 0 "
1 0 "
1 0 "
1 0 "
1 0 "
VERO 1 0 "
1 0 "
1 0 "
1 0 "
1 0 "
Ghi chii: 5 logi ti bdo six dyng trong chudn dg du</c ky hi^u: (I) LT: Te bdo linh hodn ciru sa cap;
(2) GT: Te bdo linh hodn de sa cdp; (3) GK: Te bdo thgn de sa cap; (4) LK: Te bdo thgn ciru sa cdp;
vd (5) Vero: Te bdo thgn khi xanh chdu Phi (vero). Chi so TCID50 du^c tinh trung binh cho 3 ldn Igp Igi thi nghidm.
Qua kdt qua bang 4, n h ^ xet vd TCID50 theo tiing logi tl bao thich iing cho thay
- Ddi vdi te bdo tinh hodn so cap, chiing Vn07Nt4 cho hidu gid vimt cao nhdt 10^-Vml (GT) vd lO^Vml (LT), tidp theo Id hidu gia vimt cua chiing Vn07Ntl (10^-VmI vd lO'Vml) va ciia chung Vn08Kh9 (10^" /ml vd lO^Vml).
Kdt qud chudn dp TCID50 cua 2 chung vimt VnOSKhlO vd Vn08Py7 frdn tl bdo GT vd LT la thdp nhit: Vn08KhlO (10''Vml vd 10"/ml) vd Vn08Py7 (10*Vml vd lO'Vml).
Ddi vdi td bdo than so d p , hidu gia TCID50 cao nhit Id thdp hon so vdi TCID50 frdn td bdo tinh hodn so d p (iO*"/ml frdn tl bdo thdn cim) thip ban gin 10 lln (10*Vml frdn tl bdo tinh hodn cuu).
Chung Vn07Ntl cho hidu gid vimt cao so vdi chiing Vn07Nt4 (ngupc Igi so vdi kdt qud chuan dp cua 2 chung ndy frdn td bdo tinh hoan). Thu ty ciia cdc chiing ed chi sd TCID50 trung binh tu cao den thip chuan dp frdn te bdo thdn de Id Vn07Ntl, Vn08KhlO, Vn08Py7 (diu Id IC-Vml), Vn07Nt4 (I0*-Vml), vd Vn08Kh9 (lO^'Vml); frdn te bao thdn cim Id: VnOTNtl vd Vn07Nt4 (lO'-Vml), Vn08Kh9 (lO^Vml), VnOSKhlO (lO'Vml) vd thip nhit Vn08Py7 (10^'Vml).
Doi vdi cdc chiing thich iing vd chudn dp frdn tl bdo vero, hidu gid vimt TCID50 cao vupt frpi so vdi cdc tl bdo so d p tinh hodn vd thdn, chiing VnOTNtl cho TCID50 cao nhit lO^Vml, kl tilp iln lupt Id cdc chung Vn07Nt4 (10"/
ml), Vn08Kh9 (lO-^Vml), VnOSKhlO (lO^^/ml) vd thip nhit Id chung Vn08Py7 cd TC1D50 Id lO^Vml.
- v l chi sd TCID50 ciia mdi chiing vimt theo cac logi tl bdo khdc nhau cho thay: TCID50 dgt cao nhdt khi thich iing fren tl bdo vero, kd den tl bao LT, GT, tl bao LK vd GK. Thi dy ddi vdi chung vimt dau dd Vn07Ntl, frdn tl bdo vero cho hidu gid cao nhit (10*''/ml), kl do frdn tl bdo LT (10=Vml), tl bdo GT (10"/ml), tl bdo LK (lO'Vml) vd thip nhdt d tl bao GK (lO'Vml).
Nhdn xdt frdn Id tuong ty ddi vdi cdc chung vimt khdc (sai sd cd thl cd la nhd, khdng du y nghia thdng kd). Nhu vay, xet vd khd ndng nhan ldn cua vimt ddu dd tai Vidt Nam, sau khi da thich iing, tl bdo vero cho hidu gia cao nhit, kd dd din nhdm cdc tl bao so d p tinh hodn cim va de vd thap hon Id nhdm ede td bdo so d p thgn cim vd de.
- Nhgn xdt vl khd ndng nhdn ldn cua vimt d t l bdo vero dudng nhu frdi ngupc vdi nhgn xet v l tinh cdm thy cao nhat d te bao tinh hodn so d p . Sy khac bidt ndy cd thl do tinh chit phat
KHOA HOC KY THUAT T H U Y TAP XIX S6 7 - 2012
triln cua 2 logi tl bdo trdn nudi d y dan Idp Id khdc nhau, din den s6 lupng tl bdo nhicm virut ed trong 1 chai tl bdo Id khdc nhau. Thyc v§y, kilm chiing s6 lupng te bdo d chai nudi T25, dgt dd phu dTa 100%, ciing thdi gian nudi, s6 lupng te bdo vero dgt xlp xi 10 lln cao hon tl bdo tinh hodn ciru so d p (khdng din s6 lipu)
- Kit hpp cd hai kit qua phdn tich vc d$c tinh thich nghi vd kilm tra ddnh gid hidu gid TCID50 ciia vimt sau qud trinh thich nghi cho phdp ket ludn:
Nhdm cdc le bdo tinh hodn so cap ciia dd vd cim Id nhO-ng tl bdo cd tinh edm thy cao nhit, su dyng cd hidu qud trong phdn l^p vd nhdn vimt dgu dd phdn l^p tgi Vidt Nam.
Td bdo vero, it cdm thy hon vd sir dyng khdng cd hipu qua trong phdn l^p lln diu, nhung do ddc tinh phdt triln rdt tdt, cho phdp thich ling chiing vimt ddu dd phdn Igp tgi Vidt Nam, cd thd Id ddi tupng sii dyng frong thich irng vd nhdn vimt, chl khang nguydn vdi khdi lupng Idn trong sdn xudt vacxin.
Cdc te bao so cap than dd vd thgn cim cd miie cam thy tmng binh, cd the su dyng Idm ngudn tl bdo thay thl cho tl bdo tinh hodn hodc vero tuy muc dich phan lap ho$c de sdn xult khdng nguyen.
IV, K E T L U A N
- Cd 7 chung vimt dau dd nghidn ciiu dd qua phan lap tren tl bdo tinh hodn dd deu nhdn Idn dn djnh va thich iing tot tren cdc logi te bdo so cap tinh hodn cim, tl bdo so d p thgn de, th§n cim vd te bdo ddng thuan vero. Bdnh ly tl bdo do vimt gay ra xult hidn sdm vdo ngdy thir 4 vd thti 5 sau gay nhilm d cdc lln tilp ddi thii 6 vd thii 7 tiiy theo logi t l bdo.
- Hidu gid vimt (TCID50) ciia 5 chung vimt dai dien frdn 4 loai t l bdo thich nghi (GT, LT, GK, LK) sau 7 ldn tiep ddi dao dpng frong khoang 4,4 din 6,4 Iog[TCID50]/ml tiiy thupc vdo chung phan ldp vd logi tl bdo gay nhidm.
TAI HfUTHAM KHAO
1. Trin Thj Lidn (2008), "Kit qud nghidn cthi sdn xult vd thii nghi$m vacxin vd hogtphing b$nh d^u dd". Tgp chi khoa hpc ^ thugt thi y, s6 5, tr. 93-94.
2. Adiakha S. C , Bansal M. R, Malik B, S.
(1971), Studies on sheep and goat-pox vimses. 1. Adaptation of the vimses In laboratory animals and tissue culture. Indian JAnimSci; 41: 171-175.
3. Babiuk S., Parkyn G., Copps J., Larence JE., Sahara ML, Bowden TR, Boyle D B., Kitching RP. (2007), Evaluation of an ovine testis cell line (0A3.Ts) for propagation of capripoxvims isolates and development of an immunostaining technique for viral plaque visualization. J. Vet.Diagn.Invest; 19:
486-491.
4. Bhanuprakash V, Indrani BK., Moorthy ARS., Krishnappa G. (2003), Isolation, purification and comparison of protein profiles of sheep poxvimses. Indian J Comp Microbiol Immunol Infect Dis; 24: 15-20.
5. Hosamani M., Nandi S., Mondal B., Singh R K., Rasool TJ., Bandyopadhyay S. K.
(2004), A Vero cell-attenuated Goatpox vims provides protection against vimlent virus challenge, ^c/a Virvl; 48:15-21.
6. Ireland D. C , Binepal Y. S. (1998), Improved detection of capripoxvims in biopsy samples by PCR. J rirol.Methods; 74:1-7.
7. Joshi R K., Chandra R., Rao V D R, Garg S K. (1994), Cytopathogenicity of goatpox vims. Indian J Comp Microbiol Immuno Infec Dis; 15:35-36.
8. Pandey AK , Malik BS, Bansal MR (1969), Studies on sheep pox vims. IL Immune and antibody response with cell culture adapted vims. Indian Vet.J.; 46:1017-1023.
9. Ramesh K.G. (1980), Immunological shidies on the Ranipet sfram of sheep poxvirus propagated in lamb testes cell culture. MVSc
KHOA HQC KY THUAT T H U Y TAP XIX S6 7 - 2012
Thesis.Uni Agric Sci, Bangalore, Kamataka, 13.Yogisharadhya R, Bhanuprakash
India. V,
Hosamani M, Venkatesan G, Balamumgan lO.Ramisse J, Asso J, Hassani A, Anane O, ^' ^^""^ ^^' Bhanot V, Prabhu M, Singh Jemli J. (1978), Culhire du vims claveleux ^^- (^011), Comparative efficacy of live sur cellules: Application d la vaccination et replicating sheeppox vaccine strains in au confrdle de I'lmmunite Rev.Elev.Med. ovmes. Biologicals; 39: 417-423.
Vet.PaysTrop; 31:11-19. M.Zhou J.S., Ma H.L., Guo Q.S. (2004), lI.SinghB., Rai A. (1991), Virion polypeptides Culturing of ovine testicular cells and of sheep poxvims. Indian Vet Med J ; 15: observation of pathological changes of the 239-240. ' *^^" inoculated with attenuated sheep pox ,-^ „ J n , . ^, , ^'•^s. Chinese J Vet Sci Technol; 34. 71-74.
12.Ved Prakash., Chandra R., Rao V.D.P. Garg
S.K. (1994), Cultivation of goat pox vims in Nhdn ngdy: 19-8-2012 established cell line. Indian J Virol 10; 60-63. p^an bidn- 25-8-2012
CAC LOAI RAN HOANG DA L U U GICT VIRUT GAY B $ N H Theo mpt nghien cim dupe thyc hidn tai Rimg qudc gia Tuskegee, Alabama, My, cac lodi rln hoang da Id noi luu giit vimt gay viem nao vd day cpt sdng d dpng vat. Vimt nay sau do se dupe loai muoi lan tmyen sang cdc dpng vat khac.
Nha nghidn ciiu Thomas R. Unnasch thupc Chuong trinh Nghien ciiu cdc benh tmyIn nhidm toan cdu, Dai hpc South Florida cho bill: Nghien ciiu chi ra loai rdn mang loai vimt sing tir miia nay sang miia khac.
EEEV la loai vimt gay tir vong cao d cdc dpng vat bi nhidm va cd con ngudi. EEEV dupe phat hidn tgi khu vyc Tmng, Nam vd Bdc My, dpc cac bd bidn Dai Tay Ducmg, Michigan vd Ohio.
EEEV hi lay tmydn thdng qua dudng muoi d n . Vimt ndy cd thl lay nhidm cho nhilu nhdm dpng vat nhu chim, bd sat, luong cu va dpng vat co vii. Khi nhilm vimt, ddi tupng bi anh hudng ndng nhit la con ngudi vd lodi ngya. 90% ngya bi nhidm vimt ndy sd chet. Mac du da co vacxin phdng chdng bdnh vimt nay, hdng frdm con ngya tai My vln khdng dupe tidm vacxin.Theo Bp Ndng nghidp My, tmng binh hang nam co 200 eon ngya bi nhiem vimt nay frong vdng 5 ndm qua. CJ ngudi, 35% sd ngudi nhidm vimt se td vong, 35% chju cac tdn thuong nghidm trpng Idu dai ve thdn kinh. Hidn tai chua cd vacxin phdng bdnh cho ngudi.
Cdc khu vyc ddm lay tgi vung ddng bdc Id moi tmdng cd nhieu vimt EEEV. De thyc hidn nghien ciiu ndy, cac nha nghidn cim dd lay cdc mdu mau rdn tai Rirng quic gia Tuskegee ncri vimt nay tdn tai tmng nhieu ndm qua. Kdt qua cho thiy cac con ran, chii yeu Id rdn hd mang mang vimt frong mau frong suot thdi gian ngu ddng. Vimt trong co thl rdn phdt tridn manh nhat vdo thang 4 va thdng 9 vd didu nay ly gidi tgi sao ty I? dpng vat bi nhiem vimt ndy ludn d miic rat cao vao thdng 9. Ket qua nghien ciiu se giiip con ngudi tim ra cac gidi phdp can thidp kjp thdi tranh tinh trang vimt lay lan.
NMT (Theo physorg)
