KHOA HOC KY THUAT THU Y - TAP XVII - SO 6 - 2010
KET QUA BUCJC D A U XAC OjNH TINH SINH MI§N DjCH CUA ACTINOBACILLUS PLEUROPNEUMONIAE PHAN LAP TU* LON
MOT SOTiNH PHIA BAC VIET NAM
Nguyen Thi Thu Hiing', Do Ngoc Thuy', Nguyen Thuy Duyen1,
Nguyen Viet Chirring2 , Cii Hu'u Phu1, Trudng Van Dung' TOM TAT
Budc dau xac djnh cac khang nguyen td hdp trong vacxin thd nghiem co kha nang kich thich san sinh dap dng miin dich tren Idn thuc nghiem kha tot. Tai thdi diem 70 ngay sau tiem vacxin thii nghiem, ty le Idn co khang the khang lai serotyp 2 la 75%; serotyp 5 (62,5%) va ca hai serotyp 2 va 5 (37,5%). Tai cac thdi diem khac nhau sau tiem vacxin thd nghiem:
42, 56, 70 ngay, ty le soToii co dap dng mien dich khac nhau, tuy nhien 0 mdc do cho phep.
Tii khoa: Tinh hinh mien dich, Actinobacillus pleuropneumoniae, tinh mien Bac Viet Nam.
Initial results of determining immunogenic characteristics of
Actinobacillus pleuropneumoniae isolated from some Northern provinces of Vietnam
Nguyen Thi Thu Hang, D6 Ngoc Thuy, Nguyln Thuy Duyen, Nguyen Viet Chuong , Cu HuU Phu. TrUdng V3n Dung SUMMARY
Initial identification of combined antigens m vaccines which can stimulate the production of immune response in pigs experimentally quite well. At the time of 70 days after vaccination the rate of pigs tested had antibodies against scrotyp 2 is 75%: serotyp 5 (62.5%) and both serotyp 2, 5 (37.5%). At different times after vaccination experiment (42, 56, 70 days) the proportion of pigs with immune response was different but at the non- significant level.
Key words: Immunogenic characteristics, Actinobacillus pleuropneumoniae, Northern provinces of Vietnam
I. DAT VAN DE
Benh viem phoi va miing phoi (VPMP) la benh truyen nhiem d Idn do A.
pleuropneumoniae gay ra va linn thiet hai kinh te Idn cho nghimh chan nuoi Idn cong
nghiep. Benh co ty le nhiem cao (15-39% so' Idn trong dan), lay lan rong, ty le chet co the tdi 30% tham chi 50% (Eastauhg, 2002) [2], Benh xay ra d hau het cac nUdc tren the gidi nhu chau Au. mot phan d My. Canada.
Mexico, Nam My, Nhat Ban. Han Qudc, Uc... [5].
1 Vien (hu y 2XI nghiep giong gia sue. gia ciim Thuan Thanh - BSC Ninh
De kiem soat dich benh viem phdi va mang phoi chu yeu diing thuoc khang sinh, vacxin va cac bien phap cham sdc. Sd dung khang sinh rat co hieu qua trong cac 6 dich. Co the dung khang sinh lien tuc hay gian doan de ngan chan benh, song dieu tri nhii vay se gop phan kich thich va lam tang tinh khang khang sinh hay 'on dii khang sinh trong thit gia sue giet mo. Vi vay vacxin phong benh viem phoi va mang phdi da va dang dupe nghien cUu di sau va phat trien [4].
Vdi muc tieu phong benh VPMP cho cac dan Idn trong nUdc vdi cac serotyp gay benh dien hinh phd bie'n tai mot so' tinh phia Bdc, ddng thdi chu dpng trong cdng tac phdng benh cung gdp phan linn giam gia thanh vacxin phong benh do nhap khau, chiing toi tien hanh che tao thd nghiem vacxin vd hoat keo phen td cac chung phan lap tai mpt so' tinh mien B;le Viet Nam va budc dau danh gia kha nang kich thich sinh dap dng mien dich ctia A. pleuropneumoniae phan lap.
II. NGUYEN LIEU VA PHUONG PHAP 2.1. Nguyen lieu
- Vacxin viem phoi va mang phdi vo hoat keo phen thu nghiem (vacxin TN)
- Mdi trudng hda chat cho phiin ung ELISA gian tiep
+ Hda chat cho phan dng ELISA + Khang nguyen polysaccharide tinh che td hai chiing A. pleuropneumoniae serotyp 2 vii serotyp 5 dung san xuat vacxin .
+ Khang huyet thanh khang lai serotyp 2 tham chieu va huyet thanh Ipn SPF - Nhat Ban cung cap: Khang huyet thanh khang lai serotyp 5 tham chieu - Canada cung cap:
Huyet thanh Idn ciia cac 16 dupe tiem vacxin hoac 16 do'i chdng,
+ HRP anti • pig IgG (Horse Radish Peroxidase Anti - pig IgG) Canada cung cap.
2.2. PhMng phap
+ Quy trinh che vacxin vd hoat bd trd keo phen
+ Phudng phap ELISA gian tiep
+ Xac dinh gia tri OD theo huyet thanh do'i chdng va OD cua huyet thanh kiem tra; Tinh do pha loang mau theo gia tri OD; Xac dinh ket qua ELISA theo dp pha loang cua mau theo quy trinh cua Vien nghien cuii dong vat - Queensland Uc.
+ So lieu thu dUdc trong cac thi nghiem dupe xu ly theo phudng phap thdng ke sinh vat hpc vdi sU ho trd cua phan mem Excel.
III. KET QUA VA THAO LUAN
3.1. Nghien cdu che tao thd nghiem vacxin phdng benh viem phdi va mang phdi vd hoat cd bd trd keo phen
V6i 42 chung A. pleuropneumoniae phan lap td benh pham en benh tich viem phdi va mang phdi tai 6 dia phudng nghien cuu, chUng toi tien hanh chpn 3 chung vi khudn dai dien cho 2 loai serotyp la serotyp 2 va serotyp 5a luu hanh tai mpt so' tinh mien Rdc Viet, Nam de lam khang nguyen san xuat vacxin vd hoat keo phen viem phdi va mang phdi (VPMP).
Cac chung nay thupc hai linh cd sd mau phan lap dude nhieu nhat la Hai Phdng va Thai Nguyen. Hai chung ky hieu A17- HP;
A2- HP co ngudn gdc td Hai Phong vii mpt chung d Thai Nguyen la A21-TN. Ca 3 chung deu co cac td hdp gen cd ban cua ngoai dpc to Apx duoc coi la doc td dong vai tro chinh gay benh d Idn, co doc luc cao tren chupt bach va Idn.
Do d Viet Nam chua cd tieu chuan rieng cho vacxin viem phdi va mang phdi. ni-n trong nghien cUu niiy chUng tdi da iip dung cdng thdc tinh sdludng vi khudn trong 1 lieu vacxin VPMP vo boat co bd trd keo phen theo Vien nghien cdu dpng vat - Uc vdi lieu vacxin tieu chuan cd dam dp vi khuan 4x10" CFU.
KHOA HOC KY THUAT THU Y - TAP XVII - SO 6 - 2010 V6i dam do cac canh trung (thuan khiet
100%) cua chung toi thu dUdc d hai 16 vacxin TN dat 1,65 x 10s va 1,72 x 10a, vi vay canh trung sau khi bd sung keo phen duoc xac dinh lieu tiem cho Idn la 3ml/con.
3.1.1. Ket qua kiem tra chi tieu an toan cua vacxin tren dpng vat thi nghiem
Bang 1. Ket qua kiem tra chi tieu an toan ciia vacxin tren chuot bach Sd chuot tiem, dudng va lieu tiem Lo vacxin 1 Lo vacxin 2 Ghi chu 5 chuot (0,2 ml/con, tiem phuc xoang) 5/5 chupt song
Dat 5/5 chupt sdng Dat
Cac chuot deu sdng khoe manh qua I0 ngiiy
theo doi.
5 chupt (0,5 ml/con, tiem phuc xoang) 5/5 chupt song
Dat 5/5 chupt sdng
Oat Cac chuot deu sdng khoe manh qua I0 ngiiy
theo doi.
5 chupt (0,2 ml/con, tiem tinh mach
dudi) 5/5 chupt song
Dat 5/5 chupt sdng Oat
Cac chuot deu sdng khoe manh qua I0 ngiiy
theo doi.
5 chupt ddi chUng 5/5 chupt sdng 5/5 chupt song
Cac chuot deu sdng khoe manh qua I0 ngiiy
theo doi.
Dua vao quy trinh kiem nghiem vacxin dung trong thu y cua Bp Ndng nghiep va PTNT (2006) [1], chUng toi tien hanh kiem tra cac chi tieu an toan cua hai 16 vacxin thii nghiem tren chupt bach.
Ket qua dUdc trinh bay d bang 1.
Ket qua bang 1 cho thay: Vdi cac dudng tiem va lieu tiem khac nhau, cac chupt thupc 16 thi nghiem va 16 dd'i chUng deu sdng khde manh qua thdi gian 10 ngay theo dpi. Nhu vay, ca 2 16 vacxin che thu deu dat chi tieu ve an toan 100% khi thii tren chupt bach.
3.1.2. Kit qua kiem tra chi tieu hieu luc ctia vacxin tren dpng vat thi nghiem
* Kiem tra hieu lUc tren chupt bach Ddi vdi moi 16 vacxin, chpn 10 chupt bach
khde manh, moi con tiem 0,2ml vacxin vao dudi da va 5 chupt dd'i chUng tiem 0,2 ml nude thit TYE vao dudi da. Sau 21 ngay, tien hanh thii cdng cudng dpc vdi lieu 10LD.W
(LD50 = 4,84x 107 CFU la lieu gay che't 50%
chupt thi nghiem dUdc xac dinh theo cong thUc Reed- Muench, (1938) trudc khi tien hanh cdng cudng dpc thu hieu lUc vacxin) co 10 x 4,84x 107 CFU trong 0,2 ml cho tat ca chupt thi nghiem va chupt do'i chUng. Ket qua thu dUdc trinh bay d bang 2.
Bting 2. Ket qua kiem tra hifu luc ciia vacxin tren chupt bach
Lo vacxin Sd chuot tiem (con)
Lieu tiem vacxin
(ml)
Lieu
cong (ml) Thdi gian theo doi
Sd chuot
chet (con)
chuot So sdng
Ty le bao ho (%)
TN 10 0,2 10LDM 10 ngay 1 9 90
1 DC 5 0 10LDM 18-24 gid 5 0 0
II TN 10 0,2 10LD*, 10 ngay 1 9 90
DC 5 0 10LDW 18-24 gid 5 0 0
Ket qua thu hieu luc cua hai 16 vacxin thu nghiem tren chuot bach cho thay: Hai 16 thi nghiem cho hai 16 vacxcin I, II deu co 9/10 chuot song, chiem ty le 90% so' chuot dUdc bao ho. O hai 16 dd'i chUng, chuot deu chet 100% trong thdi gian 18- 24 gid.
Theo quy trinh kiem nghiem (2006) [1], danh cho cac vacxin vi khuan vo hoat co formol va keo phen ciia bp Nong nghiep va PTNT, khi kiem tra chi tieu an toan bang phuong phap thay the tren chupt: Vacxin dUdc coi la an toan khi dpng vSt thi nghiem phai song khoe manh trong 10 ngay theo doi sau khi tiem vacxin vdi lieu sii dung. TrUdng hdp thu hieu lUc bang phUdng phap cong cUdng dpc tren dpng vat thi nghiem thay the: Vdi so' dpng vat thi nghiem dUdc cong la 5, nhom do'i chUng la 5, sau 3 tuan theo doi, chuot do'i chUng phai chet het va chupt thi nghiem sdng it nha't 3 cpn thi vacxin cd hieu luc. Nhu vay, so vdi quy dinh tren, vacxin vo hoat A.
pleuropneumoniae dam bao an toan va cd hieu luc.
3.2. Xac djnh dap Ung mien dich ciia vacxin thu* nghiem (TN)
Khang the hinh thanh sau khi tiem phdng vacxin dupe tien hanh kiem tra de danh gia dap Ung miin dich ciia Idn do'i vdi vi khuan A pleuropneumoniae co trong vacxin, dong thdi xac dinh dUdc tinh sinh miin dich ciia khang nguyen A. pleuropneumoniae trong vacxin. Theo bd' tri thi nghiem 12 Idn 5 tuan tudi dUdc chia thanh 2 16 nhu sau:
+ Ld 1 (8 Idn): tiem dudi da 3ml/con vacxin thu nghiem (vacxin TN).
Lan 1: Ldn 6 tudn tudi; Ldn 2: Ldn 7 tudn tuoi
+ Lo 2: 16 ddi chUng gdm 4 ldn.
Ldn trudc khi tiem vacxin 1 tuan (ldn 5 tudn tudi) dUdc lay mau, chat huyet thanh va kiem tra khang the bang phan ling ELISA gian tiep vdi hai khang nguyen dUdc tinh che tU hai chung thupc serotyp 2 va serotyp 5 dung che vacxin TN. Do'i chUng dUdng la khang huyet thanh tinh che dUdc gay mien dich tren ldn SPF vdi serotyp 2, serotyp 5 chuan, doi chUng am chuan la huyet thanh cua ldn SPF (do Canada va Nhat cung cap).
Sau khi xac dinh ca 12 ldn thi nghiem tai thdi diem trudc khi tiem cd khang the tu nhien trong mau dat dudi ngUdng xac dinh nghi ngd va dUdng ti'nh (ket qua xac dinh gia tri OD theo dp pha loang mau), chUng toi da tien hanh tiem vacxin TN.
Lay mau kiem tra khang the hinh thanh sau dap Ung mien dich, thdi diem tinh bat dau tU ldn tiem vacxin mui 1 (ldn 6 tudn tudi) la 0 ngay, sau dd d ldn lUdt tai cac thdi diem 42 ngay, 56 ngay, 70 ngay. Do thdi gian va kinh phi thuc hien de tai cd han, nen chung toi chi theo doi ldn dUdc den 70 ngay sau khi tiem vacxin lan 1. Cac mau mau sau khi lay, deu tien hanh chat huyet thanh de kiem tra khang the hinh thanh sau khi tiem vacxin theo phuong phap thudng quy.
3.2.1. Xdc dinh khdng the khdng S2, S5 trong huyet thanh d Ipn tiem vacxin TN sau 70 ngay
Vacxin TN dUdc phdi hdp bdi serotyp 2 (S2) va serotyp 5 (S5), do vay de xac dinh dap Ung mien dich ciia 8 ldn sau khi tiem vacxin TN, chung toi da sii dung 2 loai khang nguyen tinh che tUdng Ung de kiem tra bang ky thuat ELISA. Ket qua dUdc trinh bay d bang 3.
KHOA HOC KY THUAT THU Y - TAP XVII - SO 6 - 2010
Bang 3. Ket qua kiem tra dap Ung mien dich cua lan sau khi tiem vacxin TN d thai diem 70 ngay
STT Ky hieu tai Idn
TN KT xuat hien trong mau Idn kiem tra STT Ky hieu tai Idn
TN S2 S5 S2<5 S5
1 5 + + +
2 6 +
3 7 +
4 8 +
5 9 + + +
6 10 +
7 12 +
8 13 + + +
So con co KT/TS 6/8 5/8 3/8
Ty le % (+) xuat hien KT 75,0 62,5 37,5
KT: Khdng the; TS: Tdng so; S2: Khdng nguyen A. pleuropneumoniae serotyp 2; S5: Khdng nguyen A.
pleuropneumoniae serotyp 5; S2 & S5: Khdng nguyen serotyp 2 va 5;p (S2-S5) = 0.62;p (S2-S2&S5) = 0,51;
p(S5-S2&S5) = 0,61.
Qua ket qua bang 3 cho thay:
+ Tai thdi diem 70 ngay sau khi tiem vacxin TN, so' ldn xuat hien khang the khang serotyp 2 la 6/8 con (dat ty le 75%). Sd ldn thi nghiem cd khang the khang vdi khang nguyen serotyp 5 la 5/8 con (dat ty le 62%). So ldn co khang the khang lai ddng thdi ca hat loai khang nguyen la 3/8 con (dat ty le 37,5%). Dieu nay cho thay ca hai loai khang nguyen trong vacxin TN deu co kha nang gay dUdc mien dich cho ldn, tuy nhien d cac mUc do khac, so' ldn co khang the khang lai serotyp 2 (75%), cao hdn so'
ldn co khang the khang lai serotyp 5 (62,5%).
+ Co 3 Ipn (chiem ty le 37,5%) mang ky hieu 5, 9, 13 co khang the khang lai vdi ca hai serotyp kiem tra. Theo so' lieu theo doi cua chUng toi, thi ca 3 ldn thi nghiem nay ve tinh biet, giong khong co gi sai khac vdi cac ldn thi nghiem khac. Dieu nay cd the giai thich do cd che dap Ung mien dich cua tUng ca the co sU khac nhau.
Ket qua ve ty le xuat hien khang the trong mau ldn kiem tra sau khi tiem phong vacxin TN the hien ro tren hinh 1.
Dap ung mien dich sau tiem phong VX
Hinh 1: Ty le dap Ung mien dich ctia Ipn sau tiem vacxin TN
3.2.2. Dap ling mien dich cho Icm tiem vacxin TN tai cdc thai diem khac nhau
Xac dinh sd hinh thanh khang the d cac thdi diem sau khi tiem vacxin khac nhau, giup xac dinh dien bien khang the khang vi khuan A. pleuropneumoniae trong mau ldn ddoc tiem vacxin tai cac thdi gian khac nhau. Do vay, de xac dinh tac dong cua vacxin TN d ldn thi nghiem, chung toi da lay cac mau mau ciia ldn thi nghiem sau
%
%
%
Qua ket qua bang 4 cho thay:
+ Tat ca 8 ldn thi nghiem tiem vacxin TN tai hai thdi diem kiem tra la 42 va 56 ngay sau tiem vacxin deu co san sinh khang the khang lai khang nguyen serotyp 2 vdi ty le 100%. Tai thdi diem 70 ngay sau tiem, mile dp dap Ung mien dich giam xuo'ng (75%).
+ NgUdc lai, dap Ung miin dich vdi serotyp 2, khang the hinh thanh dap Ung do'i vdi khang nguyen serotyp 5 lai co xu hudng tang dan deu theo thdi gian tU 42 - 56 - 70 ngay sau tiem vacxin. Ty le phan tram dudng tinh lan ludt la: 12,5%; 37,5%; 62,5%.
tiem phdng vacxin TN tien hanh xac dinh khang the vdi hai khang nguyen S2 va S5 bang phan Ung ELISA gian tiep. Thong qua gia tri OD thu dUdc va khoang cho phep xac dinh vdi tUng serotyp khac nhau cung giai doan phat trien cua ldn de danh gia sU cd mat cua khang the trong mau ldn dUdc tiem phdng d cac thdi diem sau tiem vacxin khac nhau,
Ket qua trinh bay d bang 4.
5
5 + Tong hdp cac ket qua thu dupe ve dap Ung mien dich khang the ciia ldn dUdc tiem vacxin TN vdi ca hai loai khang nguyen che vacxin cho thay ty le dap Ung mien dich chung dUdng tinh vdi ca hai loai khang nguyen serotyp 2 va 5 thay co bien dpng tang dan d thdi diem 42 ngay (12,5%) den 56 ngay (37,5%) sau do ham lUdng khang the duy tri d mUc 37,5% den thdi diem 70 ngay sau tiem. Dieu nay cho thay d tai thdi diem 70 ngay sau tiem vacxin TN chi cd 37,5% so ldn thi nghiem san sinh mien dich khang lai cung mot luc ca hai loai khang nguyen co trong vacxin TN.
Ket qua thu dUdc the hien rd tren hinh 2.
Biing 4. Ket qua dap ung mien dick d lan liem vacxin TN lai cdc thdi diem khac nhau Loai KN ELISA KT d cac thdi diem tiem phong vacxin TN (n= 8)
42 56 70
S2 8/8
0/8 0/8
8/8 0/8 0/8
6/6 1/£
1/{
xuat hien KT 100 100 75
S5
1/8 2/8 5/8
3/8 1/8 4/8
5/6 0ft 3/£
xuat hien KT 12,5 37,5 62,
S2 & S5
1/8 0/8 0/8
3/8 0/8 0/8
3/£
Oil 0/£
xuat hien KT 12,5 37,5 37,
KN: Khdng nguyen; P: diMng tinh; D: Nghi ngd; N: dm tinh
KHOA HOC KY THUAT THU Y - TAP XVII - SO 6 - 2010 Tren hinh 2 cho thay d thdi diem 70 ngay
sau tiem vacxin TN thi khang the hinh thanh trong mau ldn vdi ca hai seorotyp khang nguyen tiem phong la gan tUdng dUdng nhau:
vdi serotyp 2 la 75%; serotyp 5 la 62,5% (dat tren 50%). MUc do xuat hien khang the vdi hai loai khang nguyen duy tri d mUc 37.5% nhu d thdi diem 42 ngay sau tiem vacxin.
So sanh dap irng mien djch sau khi tiem phong
P
I P
B82 • fta
• 82*88 Thdi gian sau tiem phong (ngay)
Hinh 2. Dap dng mien dich a Ipn sau tiem vacxin TN tai cdc thdi diem khde nhau IV. KET LUAN
- Vacxin che thii nghiem dat cac chi tieu ve thuan khiet (100%), an toan (100%) va hieu lUc tren chupt bach (90%).
- Vacxin TN da cd kha nang kich thich sinh mien dich do'i vdi cac ldn dUdc tiem thu nghiem d mUc dat yeu cau. Cac khang nguyen cd mat trong vacxin budc ddu kich thich san sinh duoc mien dich tren ldn thi nghiem d mUc kha tot.
Kien nghi
- Tiep tuc nghien cUu ti'nh on dinh cua khang nguyen va ty le phoi hdp hdp ly de che tao vacxin vdi dap Ung midn dich cao.
- Tien hanh cong cudng dpc, kiem tra do dai mien dich, so sanh vdi vacxin cd tren thi trUdng, xac dinh thdi gian bao quan va tien tdi Ung dung san xuat vacxin tren dien hep.
Lai cam an
Chiing tdi xin bay to long cam an chan thanh tdi: GS. P.J Blackall - Australia, GS. Y Iritani - Japan, GS. M. Gottschalk - Canada, GS. M.
Kishima vd GS. Itto - Japan vdi sU ddng gdp
nhdng y kien quy bau, cung nhitng kinh nghiem nghien cdu va rung cap nhdng nguyen vat lieu quy hiem giup chung tdi hoan thanh ditq~c thi nghiem nay.
TAI LIEU THAM KHAO
1. Cue thu y. Quy trinh kiem nghiem vacxin dung trong thu y (2006).
Http:// www.cucthuy.gov.vn I index.php.
2. M. W Eastaugh (2002), Tdng quan cac benh cua ldn. Tap chi KHKT thu y. Tap VUI, sd 3, trang 76-79.
3. Cu HQu Phu, Yoshikazu, Nguyen Thi Bich Thuy, Nguyln Thuy Duyen (2005), Tinh che khang nguyen dac hieu serotype cua A. pleuropneumoniae va mpt sd dac tinh cua chiing. Tap chi KHKT thu y, Tap XI, sol, trang 12-18.
4. Ramjeet M, Deslandes V, Goure J, Jacques M. (2008), Actinobacillus pleuropneumoniae vaccines: from bacterins to new insights into vaccination strategies. Anim Health Res Rev. Jun;9(l):25-45.
5. Taylor DJ. (1999), Actinobacillus pleuropneumoniae. In: Straw B, Taylor D, Mengeling WL, eds. Diseases of Swine.
Iowa State University Press. Ames, Iowa,
