NGHIEN CCru LIfA CHON CAC CHAT BO TRO NHO DAO CHO SAN XUAT VACXIN PHONG BENH TU HUYET TRUNG GIA CAM Sdf DUNG GIONG GOC
PASTEURELIA MUITOCIDA CHUNG N41
Hoang Vdn Khodn', Pham Thi Tuyit', Nguyin ViSt Khong', Nguyen Bd Tiip'
TOM
T A TVoi muc dich nghien curu tao vacxin dang nhfl dau phong benh tu huyet trung cho gia cam do Pasteurella multocida an toan, hieu qua, nghien curu nay nham danh gia chat lugng giong goe (P. , multocida chung N41), lira chgn moi trucmg nuoi edy vi khuan va cac chat bo Ug nhii dau phii hgp.
Ket qua nghien ciiu cho thay da xac dinh moi trudng, nong do oxy hoa tan (DO) thich hgp cho nuoi c4y vi khuan vai then gian nuoi cay trong khoang 7 den 8 gia. Hai chat bo trg: Montanide ISA TOM VG va Montanide ISA 760 VG da dugc dS xuSt six dung dua tren cac ket qua danh gia chi tieu an toan, hieu luc vacxin va phan iing ciia ga sau khi tiem chung. Cac ket qua nay la ca so cho nghien cuu hoan thien quy trinh san xuat vacxin tu huyet tning gia cam voi cac chat bo trg nhu dau va su dung giong goe Pasteurella multocida N41.
Tic khoa: Ch4t bo trg, Vacxin nhfl dau, Pasteurella multocida chung N41, Tu huyet trung gia cam,
Selection of oil emulsion adjuvants for production of fowl cholera vaccine using Pasteurella multocida
Master seed, strain N41
Hoang Van Khoan, Pham Thi Tuyet, Nguyen Viet Khong, Nguyen Ba Tiep
SUMMARY
For production of an effective and safety oi! emulsion vaccine against the fowl cholera causing by Pasteurella multocida, this study aimed at evaluating quality of the master seed (P.
multocida strain N41) selecting the suitable bacteria culture media and oil emulsion adjuvants.
The studied results indicated that a suitable medium, dissolved oxygen (DO) for bacteria culture and culture duration of 7-8 hours were determined. Two adjuvants were Montanide ISA 70M VG and Montanide ISA 760 VG proposed to use, this proposal was based on the evaluation result for safety, efficacy and response of the chicken after inoculation. This studied results can be considered as a basis for research toward completion of the oil emulsion vaccine production process against fowl cholera disease.
Key words: Adjuvant, Oil emulsion vaccine, Pasteurella multocida strain N41, Fowl cholera
I. D A T V A N D E nong, Sm (Confer, 1993). Benh co thfe do bit D- uv. u '=.^- J n M 1 J cir serotyp nao trong 16 serotyp P. multocida Benhtuhuyettrung do Pasteurella multocida , , - , • , , . j , ,-J j • „„
, „ ,, .,\ . °, •.. , • , . , , . theo phan loai Heddleston (Townsend va cs, ( r multocida) gay thiet hai Ion cho chan -,n^.^ »,r- ' . - i \--. * , •• ut,
. • ; \ . - , . , , . " , • . . , , . . - 2001). Mot trong nhung van de duac nhieu nha
nuoi gia cam, dac biet a cac nuac co khi hau • nghien ciiu va ngucri san xuat quan tam nhat 1^
phat trien vacxin an toan, co kha nang bao hp ' Phan vien thu y mien Trung
^ Vien cong nghe sinh hoc cao vai chi phi thSp, phu hgp voi diSu kien
' Vien thil y muc dau tu cua ngucri chan nuoi (Heratha va
KHOA HQC KY THUAT THU Y TAP XXI SO I - 2014
cs, 2010). Tuy nhien, nghien ciiu va san xuk vacxin song vin con gap nhiau kho khan va hien van phy thuoc vao cac vacxin vo boat va vacxin nhugc doc (Cbristensen va Bisgaard, 2000).
Cac serotyp dugc sii dung trong san xuat vacxin cho timg vung thuang dugc lira chgn dua tren nhiJTig nghien cuu dac diem dich te ciia benh (OIE, 2008).
0 Viet Nam, viec phong benh bang vacxin chua mang lai hieu qua toi uu do nhifiu nguyen nhan nhu chat lugng vacxin, quy trinh bao quan va sir dung vacxin. Hien nay, vacxin phong benh tu huyet trung gia cam da va dang duac mgt so doanh nghiep san xudt o dang bat boat vai chk bo trg keo phen, tiem 2-3 dgt/nam vai gia cam huong tning. Cac giai phap ky thuat nham nang cao chat lugng vacxin la can thiet va cap bach gop phan kiem soat dich benh tu huyet trung gia cam, dac biet la gia cim sinh san. Phan Thanh Phugng (1993) da nghien cuu phat trien vacxin tu huyet trung gia cam tir chiing cuong dgc N41 va chiing minh r5ng moi trucrng tuy tu tieu Hot- tinger phu hgp de nuoi cay vi khuan va cho dam do cao, giam lieu tiem tii 2-3ml/con xuong con 0,5ml/con, rat thich hgp cho viec tiem phong ty huyet triing gia cam dai tra. Tiep sau do, san pham k6 thita, vacxin vo hoat tir chting N41, hk hoat bang formaldehyde 0,3%, them chat bo tra keo phen do Vien thii y Quoc gia san xuat dugc phan phoi rong rai tren thi truong. Nghien cuu nay nhiim lya chgn chat bo trg nhfl dau cho quy trinh san xuat vacxin ty huyet triing sir dyng giong gdc P. multocida N4I tien toi tao san pham vacxin dam bao cac yeu cau, tieu chuan CO the SIX dyng phong ty huyet triing gia cam vai hi^u lyc va kha n5ng bao hg cao.
II. NQI DUNG, NGUYEN LIEU vA PHUONG PHAP NGHIEN cfatJ
2.1. Ngi dung nghien cihi
- Ma giong va tang cuong dgc luc cua giong qua dgng vat cam thy;
- Danh gid chk lugng ciia giong san xuat;
- Lua chgn quy trinh tao khang nguyen vi khuan P. multocida N41 voi chat lugng t6t;
- Lira chgn dugc chat bo trg nhii dau cho chat lugng vacxin tot nhat phong benh cho ga.
2.2. Nguyen lieu
Giong gdc P. multocida N41
Cdc loai moi trudfng: moi truong tong hgp cua cac hang Merck (Germany), HiMedia (India) va TiTan (An Do); Blood Agar, Sarbouro Agar, Brain Heart Broth, BHI Agar, Yeast Extract, Pepton... Cac loai huyet thanh cim, huyet thanh ht, mau cim.
Cdc loai hoa chat: Dung dich NaOH IN, H,SO^ 5%, Formaldehyde, Thimerosal, Antifoam 204.
Chat bo trff: Dau khoang Montanide ISA 50V (Seppic, Phap); Eolan, Sinofon Span 80, Sinofon Teen 85 (Total, Phap)
Mdy vd thiet bi chuyen diing gom cac loai binh Schott, Pipette va cac loai ong nghiem;
mdy len men sue khi BioStat B (dung tich 10 lit); he thong len men sue khi tu dgng Bioflo 5000 (dung tich 80 lit, NBS, Hoa Ky); thiet bi gay nhfl Ultra Turrax T50 Basic (IKA - Ditc);
May siet nut chai...
2.3. PhifOTig phap nghien cuu 2.3.1. Kiem dinh giong P.multocida N41
Ria cay vi khuan tren dia thach huyet thanh 10%, vi khuan phat trien thanh nhflng khuan lac rieng re, quan sat khuan lac ducri kinh hien vi voi do phong dai 20 lan, gdc chieu den chech 45". Dac tinh gay benh tren dgng vat cam thy dugc xac djnh bdng phuang phap gay nhiem cho ga tren 2 thang tuoi (khoi lugng ca the 0,8 - 1,2 kg), \\h\x 10LD50; theo doi ty Ie chet, thdi gian gay chet ga. Phan lap vi khuan tir mau tim ga chet tren thach mau va thach huyet thanh.
Chi so LD50 dugc xac dinh theo phuang phap CLia Reed & Miiench (1938).
2.3.2. Phuffngphdp xdc dinh ddm do vi khudn Canh khuan da pha loang theo he so 10 de dugc nong do tir 10"^ den 10"' dugc cay tren cac dia thach mau (nong do 10', IO*' va 10', 3 dia/
nong do); nudi cay d ST^C/l 8-20 gid sau do dem sd lugng khuan lac tren timg dia d cac do pha loang khac nhau va xac dinh sd lugng vi khuan CO trong I ml canh khudn ban dau theo cdng thiic X = a x N x IO(Xlasd lucmg vi khuan cd trong Iml canh khuan ban dau; a la sd khuan lac trung binh dem dugc trong lOOpl canh khuan d mdi ndng do pha loang tuong irng; N la ndng do pha loang canh khuan).
2.3.3. Phuffng phdp xdc dinh chi tieu an todn cda vacxin tren gd
Tiem dual da 2ml vacxin/con cho 5 ga do khde manh ( > 2 thang tudi, khdi lugng ca the 0,8 - 1,2kg). Vacxin dat chi tieu an toan khi ty le sdng cua ga dat 100% d ngay thir 10 sau tiem.
2.3.4. Xdc ^nh hieu luc cua vacxin tren gd qua thu thdch vdi vi khudn cuong doc.
Tiem dudi da 0,5 ml vacxin/con cholO ga do.
Ld ddi chiing gdm 10 ga dd khde manh khdng dugc Uem vacxin. Sau 28 ngay, ga thudc 2 Id dugc thu thach vdi vi khuan P. multocida chimg N41 cudng ddc vdi li^u tiem 10LD50 (tiem dudi da).
Ga dugc theo doi 7-10 ngay sau khi cdng cudng dgc. Ket qua dat yeu cau khi ga d Id ddi chiing chet 100% va phan lap dugc vi khuan P.multocida tir mau tim ga chet. Ty le bao hg vdi Id dugc tiem vacxin dua vao ty le (%) sd ga cdn sdng.
III. KET QUA NGHIEN ClTU vA THAO LUAN
3.1. Mot so chi tieu ciia gidng san xuat (vi khuan P. multocida chung N41)
Sau khi md gidng, chdt lugng gidng gdc dugc danh gid dua tren dac tinh sinh hgc va dgc luc cua vi khudn ddi vdi gia cam mdn cam. Kit qua dugc trinh bay d bang l(bdng 1).
Bang 1. Ket qua kiem djnh mot so chi tieu giong san xuat Bac tinh
Nuoi clly
Dung quang
Gay benh
Chi tieu Tren thach thu'dng Tren thach mau Mditru-ffngBH^S/g/lit) Thach huy§t thanh (10%) Dac tinh dung quang (tren thach huyet thanh -10%) LD50
Thdi gian gay ch§t vai li§u gay nhi§m 10 LD50
Ket qua danh gia Khuan lac nho, giong phat tnen Item
Khuan lac trdn, tren, bong lang mau trang due, giong phat tri4n t6t
Mdi tripdng dye nhe, co van nhu" su-ang mu Gidng phat tri^n tot, khuan lac tron, bong lang, trong suot Khuan lac co dung quang dac tru'ng: Mau xanh la, xanh la m?
chi§m phln Idn dien tich khuan lac. Dien tich khuan laccdn l@l CO mau vang da cam
180 t6 bao 3 6 - 4 8 gid
100% ga chit phan lap diFffC P.multocida N41
Ket qud bang 1 cho thdy vi khuan gidng gdc phat trien tdt nhat tren thach mau va thach huyet thanh 10%; phdt trien kem tren mdi trudng thach thudng va mdi trudng nudc thit (BHI) khong bd sung huyet thanh. Dieu nay hoan toan phii hgp vdi nhihig md ta ve dac tinh nudi cay
cua vi khuan P. multocida. Khuan lac cd hi?n tugng dung quang dien hinh; mau xanh chi^m phdn ldn dien tich khuan lac trong khi mau vang cam chiem phdn dien tich cdn lai.
Khi gay nhilm vdi ga tren 2 thang tudi, vi khudn gidng gdc the hien dgc luc cao. Chi
KHOA HQC KY THUAT THU Y TAP XXI SO 1 - 2014
tieu LD50 cua gidng la 180 te bdo. Vdi Ii6u gay nhiem 10LD50 te bao vi khuan, thdi gian gay chet ga dd la tir 36 den 48 gid sau khi gay nhiem. Vi khuan dugc phan lap tir tdt cd cac mdu mau tim ga chet. Nhu vay gong vi khuan P.
multocida chung N41 dugc bao quan ddng khd tai thdi diem nghien cuu dat tieu chuan cho cac budc nghien ciiu tiep theo
3.2. Anh hudrng ciia mdi trudng nuoi cay den kha nang tang sinh ciia vi khuan P. multocida Theo Phan Thanh Phugng (1993), mdi trudng Hottinger phu hgp de nudi cay vi khudn ty huyet triing cd dam do cao, trong dieu kien len men sue khi cd the dat 26,5 x 10^ te bao vi khuan/ml canh khuan. Hien nay, mdi trudng
nudi edy vi khuan dugc nhieu hang tren the gidi san xuat vdi nhieu uu diem. Tuy nhien, gia thanh cho mdt dan vi the tich mdi trudng nudi cay lai cao hem so vdi mdi trudng Hottinger.
De xac dinh dugc cdng thirc phdi hgp cac thanh phdn mdi trudng nudi cay phu hgp nhat ddi vdi vi khuan P. multocida chung N4I va so sdnh chdt lugng mdi tnidng cua cac nha san xudt khac nhau, chiing tdi da thii nghiem 9 cdng thiic phdi hgp mdi trudng theo hudng dan cua cac hang Mecrk (Due), Himedia (An Do) va TiTan (An Do) ( bang 2). Nguyen lieu cua mdi hang san xuat dugc phdi hgp tao 3 cdng thiic.
Ty le phdi hgp cac thanh phan trong mdt cdng thiic gidng nhau vdi 3 nhdm nguyen lieu cua 3 hdng sdn xudt.
Bang 2. Thanh phan cac moi tru'O'ng nuoi cay Thanh phan (g/lit)
Brain Heart Broth Yeast extract Meat extract Huyet thanh be
Cong thLKC 1 37,0
0 0 10%
Cong thi>c II 37,0 13,0 0 10%
Cong thu'c III 37,0 13.0 8.0 10%
Cdng thirc 1 chi sir dyng nguyen lieu la mdi trudng BHI vdi hdm lugng 37g/lit, cdng thirc II bd sung them chat chiet nam men (Yeast extract) vdi ham lugng 13g/lit va cdng thiic thir 3 dugc bd sung them 8g chat chiet thit (Meat extract) trong mdi lit mdi trudng nudi cay. Mdi trudng sau khi pha che dugc thanh trung d nhiet dd ni^cns phut, lam mat tdi nhiet do 3TC, bd
sung them 10% huyet thanh be vd triing trudc khi truyen ciing mdt the tich gidng de nudi cay.
Quy trinh tren ap dyng cho mdi cdng thiic tao mdi trudng nguyen lieu ciia ca 3 nha san xuat, tdt ca cac thi nghiem dugc deu dugc thuc hien tren he thdng nudi cay tu ddng Bioflo 5000 vdi ciing chuang trinh duac van hdnh. Ket qua dirge trinh bay d bang 3
Bang 3. D a m dp vl khuan trong cac moi t r y i n g nuoi cay
Hang san xuat Merck Himedia
Titan
Dam do vi khuan (x10'CFU/ml) Cong ttiiFC 1
26,5 ± 0.93 21,0 ±1,00 15.5 ±0,81
Cong thirc II 28.0 ± 0,94 26.0 ±0,87 19,0 ±0,94
Cdngr thi>c Ul 28.0 ± 0.93 27.5 ± 0.87 22,0 ± 0,81
Nhu vay dam dg vi khuan dat cao nhat vdi mdi tnidng pha che theo cdng thiic 111 su dung nguyen Ii?u ciia ca 3 nha san xudt, Dieu dd cho
thdy bd sung Yeast extract. Meat extract Iam tang dam do vi khuan. Dac biet, bd sung Yeast extract dan den dam do vi khuan tdng len ro r?t
K6t qua cflng cho thdy sy khac nhau ve chdt lugng mdi tnidng tir cdc ngudn nguyen lieu khac nhau. Mdi trudng vdi nguyen lieu cua Merck cho ket qud nudi edy tdt nhat, tiep den la mdi trudng Himedia va thdp nhat Id mdi trudng ciia hang TiTan Vdi mdi trudng ciia Merck, chi cdn bd sung Yeast extract dam do vi khuan da dat dugc 28,0 X 10^ te bao vi khuan/ml canh khuan, viec bd sung Meat extract cd the khdng can thiet.
Mdi trudng cua Himedia cung cho ket qua nudi edy khd tdt, dam do vi khudn dat dugc sau khi dugc bo sung day dii cac thanh phdn nguyen lieu Id 27,5 x 10^ vi khudn/ml canh khuan so vdi 28 x 10^ vi khuan/ml khi dung mdi trudng ciia Merk va 22 X 10^ vi khuan/ml khi dung mdi trudng cua TiTan. Tuy nhien, su dyng mdi trudng do hang Himedia sdn xudt cho hieu qua cao nhat vi tai thai diem thi nghiem gia thanh mdi trudng nguyen lieu cua Merck cao gap 2,5 - 3 ldn so vdi Himedia va TiTan. Trong khi dd, Himedia va TiTan cd gid thdnh tuong duong nhung hieu qua nudi cay vi khudn khi diing mdi trudng ciia Himedia cao hon nhieu so vdi TiTan. Nhu vdy sir dung mdi trudng do Himedia (An Do) san xudt la phi] hgp nhat de san xuat khdng nguyen
P. multocida chung N41 hen quy md ldn trong dieu kien hien nay.
3.3. Xac dinh chi tieu ham lu-ffng DO toi uu trong qua trinh nuoi cay vi khuan P.
multocida chung N41 bang cong nghe len men sue khi
Len men sue khi sinh hgc cho phep tao dugc canh khudn vdi dam do cao, dap img tieu chuan cao. Qua trinh len men syc khi dugc thuc hien tren he thdng thiSt bi Ur ddng, dam bao cac dieu kien nudi cay vi khudn nhu: do pH, nhiet do, ham lugng oxy hda tan (DO). Ddi vdi P.
multocida, qua trinh len men dugc thuc hiSn trong didu kien nhiet do 37''C va pH 7,2. Da tien hanh khao sdt chi tieu DO tao ca sd xay dung quy trinh len men tdt nhat.
De xac dinh dugc chi tieu nay, dnh hudng ciia miic DO khac nhau tdi dam do vi khudn cua mgt sd me Ien men dugc xac dinh (bang 4).
Dam dd vi khuan tai 4 thdi diem trong qud trinh Ien men dugc kiem tra nham ddnh gia tdc do tang sinh cua vi khuan va xac dinh thdi diem thich hgp de dimg len men. Lap lai thi nghiem 3 Ian ddi vdi mdi miic DO
Bang 4. Anh hu'O'ng cua DO den dam do vi khuan P. multocitSa va thd>i gian nuoi cay
DO (%) 40 60 70 80 90
Dam dp vi khuan (x10^CFU/ml ) tai cac thm diem 5 giiy
11.0 ±0.82 13.5 + 0.50 12.0 ± 0 , 6 6 11.0 ±0,00 10,5 ±0,66
Ggicy 15.5 + 0.66 17.0 ±0.81 23.5 + 0.66 22.0 ± 0.66 18.5 + 0,61
7 giiy 21,5 ±0.81 26.5 ± 0.50 28.5 ±0.66 30.5 ± 0.87 26.0 ± 0.82
nuoi c^y 8 giiy 22.0 ± 0,50 28,0 ± 0,50 28.5 ± 0.50 28.0 ±1.05 26.5 ± 0.50
Thi nghiem cho thay dam do vi khuan sau Ien men dat cao nhdt ( 30,5 x 10^) d miirc DO 80% tai thdi diem 7 gid sau khi truyen gidng.
Sau dd, d thdi diem 8 gid dam do vi khuan giam dl cdn 28,0 X 10^ vi khuan/ml canh khudn. Vdi miic DO thap nhat (40%), dam do vi khuan cao nhdt cflng chi dat 22 x 10^ vi khuan/ml sau 8 gid
nudi cay. Tai muc DO cao nhat (90%) dam dg vi khudn tdi da dat dugc d thdi diem sau 8 gid nudi cay la 26,5 x 10^ vi khudn/ml.
Vl khuan phdt trien, tang sinh manh nhat sau 5-7 gid nudi edy. Day Id khoang thdi gian the hien sy trao ddi chat manh me, bidu hien qua sir bien ddi mdu sac mdi trudng, sir thay ddi dp pH,
KHOA HOC KY THUAT THU Y TAP XXI SO I - 2014
miic do SU dyng O,. Vdi di£u kien dinh dudng xac djnh, vi khudn P mutocida chi tdng sinh ve sd lugng den mdt thdi diem nhdt dinh, sd lugng vi khudn sdng xac dinh duac se giam di nSu tiep tuc qua trinh len men. Nhu vdy miic DO phii hgp nhdt de nudi edy vi khudn P. multocida chiing N4I la 80%, thdi diem ket thiic len men tdt nhat la sau 7 gid . Thdi gian nudi cay khdng qua 8 gid.
3.4. Lira chgn chat bd trg phii hgp de san xudt vacxin nhu ddu phdng benh tu huyet triing cho gia cam
Chung tdi da tien hanh thu nghiem vdi 4 loai chat bd trg tao nhfl khac nhau cua 2 nha cung cap dugc biet den rdng rai la Seppic (Phdp) va Total (Phap). Khdo sat danh gid chdt lugng vacxin thdng qua cac chi tieu cam quan, hda Iy, an toan va hieu luc. Ket qua duac the hien tai bang 4, 5 va 6.
Bang 4. Ket qua danh gia cam quan vacxin chat lu'o'ng nhu dau thii' nghiem
Chat bo tro*
lylontanide ISA50V2 Montanide ISA 70 MVG
Montanide ISA 760 VG
Eolan 130 T / l e
50 (V/V)
70 (WW)
70 (W/W)
60 (VA/)
Chi tieu danh gia
(%)
Trang nga TrIng sOa Trlng siJa TrSng s&a
Kich thw&c hatnhu (tJlicron)
<1,3
<1,0 S1,0
^1,3
Drop test
++++
++++
++++
+++
Do nh&t (Viscosity) o2S>C (mPa.s) 100
35 40 60
Do on dinh
& nhiet do phong
> 30 ngay
> 30 ngay
> 30 ngay
< 30 ngay
Ca 4 loai chat bd trg gdy nhu theo hudng dan ciia nha san xuat deu cd kha nang tao hat nhii tdt, mau sdc va tinh chat vacxin phu hgp theo yeu cdu. Tuy nhien, vdi cac thiet bi kiem tra chuyen dung cho thay Eolan 130 cho kich thudc hat nhfl ldn nhat (> 1,3 micron); Monta- nide ISA 70 MVG va Montanide ISA 760 VG deu cho kich thudc hat nhii < 1,0 micron. Mon- tanide ISA 50 V2 cho kich thudc hat 1,0 - 1,3 micron. Theo tieu chuan ciia nha san xuat, kich thudc hat nhu < 1,0 micron se cho chdt lugng vacxin tdt nhdt.
Montanide ISA 50 V2 cho vacxin cd do nhdt cao nhdt (100 mPa.s), ti^p ddn la Eolan 130 (60 mPa s). dg nhdt thap ban la Montanide ISA 760 VG va thap nhdt la Montanide ISA 70 MVG (35 mPa.s). Vacxin cd dg nhdt thap duac cho la dl tiem va it gay phan iing eye bd.
6 nhiet do phdng, vacxin sir dung Eolan 130 cd do dn dinh thdp nhdt. Sau 30 ngay da cd hien tugng tach Idp Trong khi dd, vacxin su dung 3 loai chat bd trg cdn lai deu cd do dn dinh cao hon trong cimg dieu kien bdo quan.
Nhu vdy Montanide ISA 70 MVG va Montanide ISA 760 VG la 2 chat bd trg cd uu diem thdng qua danh gia tinh chat ly hda.
Vdi lieu tiem 2 ml/con, tdt ca cac Id ga deu cd ty le nudi sdng dat 100% trong thdi gian theo ddi 10 ngay sau khi dugc tiem vacxin. Dieu dd cho thay ca 4 loai chat bd trg sii dung de che vacxin phdng ty huyet tning cho gia cdm deu an todn ddi vdi ga tren 2 thdng tudi.
Chi tieu hieu luc ciia 4 Id vacxin dugc xac dinh bang phucmg phap thu' thach vdi vi khuan cudng ddc ddi vdi ga dugc tiem vacxin sau 28
ngay. Ket qua cho thay: Ld su dyng vacxin vdi chdt bd trg Montanide ISA 760 VG cd ty le bdo hg cao nhat, dat 86,66%; tiep den la Id sii
dung Montanide ISA 70M VG (ty le bdo hd dat 80,00%).
Bang 5. Anh hu'O'ng cua chat bo trcr den chi tieu an toan va hieu lire ciJa vacxin tu huylt triing gia cam nhu dau
Bo trp*
Montanide ISA 50 V2 Montanide 1SA70M VG
Montanide ISA 760 VG
Eolan 130
An toan Lieu tiem
(ml/con) 2,0 2,0 2,0 2.0
Soga tiem 5 5
5
Ty le song
100 100 100 100
Hi^u LLFC Lieu tiem
(mt/con) 0,5 0,5 0.6 0.5
So ga tiem
10 10 10 10
Ty te bao tio
(%)
63,33 80,00 86.66 36.66
Nhu vay, vdi ty le bao hd dat tren 80%, Montanide ISA 70M VG, vd Montanide ISA 760 VG la hai chat bd trg phii hgp nhdt cd the dugc lua chon de che tao vacxin phdng benh ty huyet trung cho gia cam. Tuy nhien, lua chgn
chat bd trg cdn phai xet den nhimg phan iing bat Igi ma vacxin gdy ra ddi vdi sit sinh trudng va phat trien cua ddi tugng gia cam dugc tiem. Do dd cac bieu hien phdn ung eye bd, trang thai site khde dugc theo ddi va tdng hgp d bang 6.
Bang 6. Ket qua theo doi phan u'ng cCia ga sau khi du'o'c tiem vacxin tu huyet trung vo-i chat bo tro- khac nhau (n=50)
Loai dau
Montanide ISA 50 V Montanide ISA 70M VG
Montanide ISA 760VG
Eolan 130
Phan U'ng cue bp Bieu hien cue bp Sifng keo dai, khd
tieu tan Sung nhe, h§t sau 5-
7 ngay
Su-ng to, tang smh t l bao qua mLrctai vi tri tiem, keo dai nhi4u
ngay.
So con 18
24 Tyle
(%)
36,00 14,00 0,00
48,00
Phan U'ng toan than Bieu hien
Hai met mdi, giam an, giam van dpng Giam an a ngay thu'
3-5 sau khi tiSm
Giam an ro ret. gi^m van ddng, sut can...
So con
22 T^l^
(%)
16,66
73,33
Phdn ling cue bd de nhdn ra nhdt la tang sinh td chiic, sung keo dai tai vi tri tiem ddi vdi Id thi nghiem diing chdt bd trg Eolan 130. Vdi Id nay, sau 3-5 ngay dugc tiem vacxin, ga bit ddu cd nhiing bieu hien met mdi, di lai cham chap.
gidm an, sut cdn. Nhiing bieu hien tren chi gidm dan va mdt di khoang 45 ngdy sau tiem. Nhflng bieu hien tuong tu dugc ghi nhdn vdi Id ga dugc dem vacxin sir dung Mmotanide ISA 50V lam chdt bd trg. Tuy nhien, miic do bidu hien nh?
KHOA HOC KY THUAT THU Y TAP XXI SO 1 - 2014
han, ty I? thap hem va thdi gian gay phan iing ngdn han nhieu, gd nhanh chdng phuc hdi siic khde. Ddi vdi 2 Id ga dugc tiem vacxin sir dung chdt bd trg Montanide ISA 70M VG va Monta- nide ISA 760 VG khdng ghi nhan nhflmg phan ling bat thudng sau khi dugc tiem. Tai vi ui tiem cd the quan sat thay vacxin tdn tai trong thdi gian song khdng gay phdn iimg viem hoac sung qua miic, ga cd the bai met mdi sau khi dugc tiem vacxin rdi phue hdi rat nhanh sau dd.
Ket qud theo ddi cho thdy Montanide ISA 70M VG vd Montanide ISA 760 VG la hai chdt bd trg phii hgp nhat cd the dugc lua chgn cho cac nghien cuu tiep theo tidn tdi hoan thanh quy trinh san xuat vacxin phdng benh ty huyet trung cho gia cam nhfl dau.
IV. K E T L U A N
Gdng vi khudn P. multocida chiing N41 dirge bao quan ddng khd dat cac tiSu chuan cho cac budc nghien ciiu sdn xudt vacxin thdng qua danh gid cdc chi tieu kiem tra kha nang phat trien tren cac moi trudng nudi edy, dac tinh dung quang va dOc luc.
Sir dyng cac nguyen lieu cua hang Himedia (An Dg) trong pha che mdi trudng nudi cay vi khudn gidng gde vua dam bdo kha nang phat tnen ciia vi khudn, vua cd hieu qua kinh te, phii hgp vdi dieu kien Viet Nam.
Ndng dd oxy hda tan (chi sd DO) phu hgp cho qua trinh phdt trien cua vi khudn gidng gdc la 80% va thdi gian nudi edy khdng qua 8 gid.
Dd xdc dinh dugc hai chdt bd trg Montanide ISA 70M VG va Montanide ISA 760 VG phii hgp cho san xudt vacxin ty huyet triing keo phen vdi gidng gdc P. muhocida N41.
Cac nghien cmi tiep theo nham sdn xuat thu nghiem va khao nghiem vacxin tu huygt triing nhii dau vdi gdc N4! can dugc thuc hien.
T A I L I E U T H A M K H A O 1. Confer A. W. 1993. Immunogens of Pasteu-
rella. Veterinary Microbiology 37:353-368.
2. Cbristensen JP and Bisgaard. Fowl cholera.
Rev.sci tech.off.Int.Epiz.,2000,19(2), 626-637 3. Heratha C , Kumarb P., Singhc M., Kumard
D., Ramakrishnane S., Goswamic T.K., Singhf A, Ramc G.C. (2010). Experimental iron-inactivated P. multocida A: I vaccine adjuvanted with bactenal DNA is safe and protects chickens from fowl cholera.
Vaccine 28 (11). 2284-2289
4. OIE (2008) Fowl Cholera. OIE Terrestrial Manual Chapter 2.3.9., 524-530 5. Phan Thanh Phugng (1993) Te bdo T va B
- Lympho d ga dirge mien dich bdng vacxin tu huyet triing cd cac bd trg khac nhau. Tap san cac cdng trinh nghien ciiu Khoa hgc ky thudt Vien Thu y Qudc gia. Nha xuat ban Ndng nghiep, tr.48 - 53.
6. Townsend K M., Boyce J.D., Chung J.Y., Frost A., Adler B. (2001) Genetic organization of Pasteurella multocida cap Loci and development of a multiplex capsular PCR typing system. Journal of Clinical Microbiology 39 (3). 924-929 Nhdn ngay 31.8.2013
Phan bien ngdy 20.10.2013
