KHOA Hpc C 6 H G N C H |
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KHAIMG VI RUT GAY HOI CHIJIMG ROI LOAM 5II\H SAM VA Hd H A P d LOIM
VQ T i d n L i m ' , P h ? m C d n g Hojlr', P h ^ m Tlii T i m '
T6MTAT
04 chimg PRRSV p h i n Up dupe tir Ipn m i c bdnh tiu xanh 4 Hung Yen, Ddng Nai, Ha N<ii, -Vinh Phuc ky hifu PRRSV PR-HY 01, PR-DN 01, PR-HN 01 va PR-VP 01 c6 TClDj, tuong mig l i 1 0 " , 10*', 10*", 1 0 " . Trong do, chung d?! didn k h i n g nguydn l i PR-HY 01, chung vi nit nay bj b^t boat bdi formalin 0,5%. Khing the trong ldng do ti-iing g i dupe gay midn dich bdi chiing PR-HY 01 dat cao nhit o lu.in thu 11 nhung c6 kha nang ti^uig hda PRRSV cudng dOc tu tuin thii 4, Khing thd trung hda t6n luu 1 th6ng trong Ipn, vi viy dinh ky hang tiling tidm n h i c lai k h i n g thd thy dpng dd c6 hieu qua phong bdnh tdt nhiit
Tir k h o i Ldng do trung, khing tlii diu dfng, PRRSV.
Bdnh tai x a n h h a y h d i c h i i n g r d i logn s i n h s i n v i hd h a p d Ipn {Porcine Reproductive and Respiratory Syndrome - PRRS) l i mOt b e n h t r u y e n n h i e m n g u y hidm e u a Ipn m p i n d i g i d n g , m p i liia tudi, g a y thidt hai kinh t e e h o n g a n h c d n g n g h i d p c h i n nudi Ipn tidn k h i p t h d gidi. T a i Vidt N a m , n a m 1997, k i e m t r a huydt t h a n h h p c e h o b i e t 1 0 / 5 1 Ion g i d n g n h a p tii My cd h u y e t t h a n h d u o n g t i n h vdi vi n i t g i y hdi chiing rdi loan s i n h s a n v i h d h i p (PRRSV). T u y nhidn, s u b i m g p h i t t h i n h d i c h v i g a y t d n t h i t d a n g b i o d d n g c h o n g i n h c h i n n u d i Ipn b i t d i u v i o t h i n g 3 n i m 2007.
Hidn tai, trdn t h e gidi e d n h i d u loai v i e x i n p h d n g bdnh tai x a n h b a o g o m c i e v i e x i n vd h o a t n h u Progressis, S u v a x y n P R R S , h i g e l v a e P R R S KV, Suipravae PRRS h a y c i e v i e x i n s d n g n h u o c d d c n h u Amervae PRRS, P y r s v a c 183, P o r c i h s P R R S , Ingelvae PRRS MLV. T u y n h i d n , h i e n tai n h i i n g v i e x m n i y van k h d n g d a p l i n g d u p e y d u c a u vi k i e m s o a t v i phong c h d n g P R R S h-dn t h e gidi. T a i hOi ttiio c h u y d n ' dd vd ti-idn v p n g c i i t h i d n h i d u q u i ciia v i e x i n c h d n g PRRS d u p e td c h t t e t a i M y n i m 2007, c i c n h i k h o a hoc ttidng n h i t q u a n d i d m r i n g s d p h i i m a t k h o a n g 5-10 n i m niia d d c d t h d c d q u y h i n h c h d t a o v i e x i n .tidu c h u i n U-dn t h d gidi. N h u v i y , t i n h d e n h i d n tai, c i c n u d c v a n p h a i t u i p d u n g m o i b i d n p h i p d e k i e m soat PRRS c h o d i n Ipn eila n u d c m i n h m i k h d n g ttid trdng c h d v i o v i e x m d u p e s i n x u i t tir e i e n u d c k h i e . Trong n g h i d n eiiu n a y , k h i n g ttid ttt^ dOng k h i n g
PRRSV d u p e s a n x u a t n h i m g d p t h d m m d t g i i i p h i p p h d n g ti-j b d n h tai x a n h trdn d i n Ipn d Vidt N a m .
L Btil TUDNG Vi PmniMi PHAP NGHEN CUU 1. D d i tirpng
04 c h i i n g VI n i t g a y b d n h tai x a n h p h a n l i p d u o c d Viet N a m k y hipu l i PR-HY 0 1 , PR-DN 0 1 , PR-HN 01 v i PR-VP 0 1 .
2. P h u o n g p h i p n g h i d n eiiu a. Nudi cay vi nit tien tibio
PRRSV dupe nudi bing td bao MARC 145. Tnrde khi su dung, td bio dupe hoat hda Uong chal nudi ciy vdi mdi ti-udng MEM cd bd sung 5% huydt ttianh bao tiiai bd (FCS). Te bio dupe nudi Uong tii am d nhidt dd 37''C, 5% CO; ti-ong 3 ngiy de dat mat dp Vf ti bio/ntt.
Cic chiing vi nit dupe lay nhidm trdn td bio MARC 145 da boat hda. Chai ddi ehiing chi cd te bio GS va mdi UTrdng DMEM cd bd sung 5% FCS.
Theo ddi hidu img buy hoai te bio (CPE- cytopattne effect) mdi ngiy. Khi CPE d?t khoang 90- 95% Uiu dich vi nit khdi chai nudi ciy rdi ly tim fliu dich ndi cd ehiia vi nit.
b Xac dinh liiu TCIDso
C h u i n d p vi n i t d u p e t h u c h i d n ti-en dia n h u a 96 g i d n g n u d i td b i o M A R C 145.
D i c h vi n i t d u p e Hen h i n h p h a l o i n g t h e o h e s d 10 ti-ong m d i U u d n g H a n k ' s r d i b d s u n g vao c i c g i d n g te b a o . Vi n i t d u p e h a p p h u v a o td b i o t r o n g 2 gid.
T i d p h i e n u d i td b i o b i n g M E M c d bd s u n g 5% F C S . ' Cong ty Cd phan Phat tridn Cong ngh? Ndng thon
' Bp Khoa hpc va Cdng nghd ' Vidn Dai hoc M d Ha Npi
' N 6 N G NGHIpP VA PHAT TRIEN N 6 N G T H 6 N - KV 1 - THANG 10/2012 65
KHOA HQC C 6 N e N G H t
Quan sit hidn CPE ti-ong vdng 5 ngiy dd xic dinh TCIDso theo phuong phip cila Reed vi Muench (1938).
c. Phuong phip giy bit ho^tvinit
PRRSV dupe bit hoiit bdi formalin vdi 3 ndng dO:
0,1«, 0,391, 0,5% d nhidt do 37X ti-ong 12 h. Sau 12 h tnmg hda formalin bing phdt phit naUi, thpc hidn 1.1c djch lidn tijc ti-ong 24 gid.
d Phuong phip EUSA
Phan ling dupe thuc hidn vdi khing thd pha loing trong dung djch ddm cacbonat, mdi gidng phii 100 111 rdi li qua ddm d nhidt dd 4"C; djch khing nguydn pha loang ttieo ty Id 1/100 Uong PBS, u d nhidt dp 37»C/ 1 gid; goat anti-rabbit IgG HRPO (Sigma) pha loing 1/10.000, Cl d nhidt dO phdng
Bing 1. Kdt qui giy nhidm
trong 45 phiit; cp chit sinh miu TMB u ti-ong 15 phiit A 37»C vi dimg phin iing bing HiSO, IM. Dgc kdt qui phin ling trdn miy dpe dia ELISA, buoc sdng 450 nm.
1 . KfrWlANGUlCNCtlU
1. Xic dtnh Udu TCIDa, ciia PRRSV ti<n td bij min cim
Lidu TCID,5o eua 4 ehiing PRRSV flii nghidm dupe xic dinh dya trdn mirc dd hiiy hoai td bao MARC 145 sau 5 ngiy giy nhidm. Muc dich cila vide xic djnh TCIDK li dd xie djnh lidu khing nguySn phuc vy giy dip ting midn djch Udn dOng vit thi nghidm, ddng flldi lira chpn chiing cudng dpc de dinh gii khi ning tiling hda vi nit cua khing flid ch^
t^o dupe. Kdt qui dupe tiinh biy ti-ong bing 1. ' vi nit ti-dn td bio MARC 145
TT 1 2 3 4
Kyhidu mau PR-HY 01 PR-DN 01 PR-HN 01 PR-VP 01
CPE theo cac dd pha loing vl nit (%) 10'
100 100 100 95
10' 100 100 100 95
10' 98 95 95 80
lO*
85 80 80 70
1 0 •>
80 75 65 60
10<
70 60 60 52
10' 50 48 48 35
10*
30 35 25 20
10' 10 10 10 5
TCIDs, 10«
10«
1 0 "
10"
So lieu thu duoc trong bang 1 cho thay, cac chung PRRSV mac dil dupe phan lap tir cac dia phuong khac nhau nhung dpc lire la tuong duong nhau.
De phuc vu che tao khang th^ thu dong qua long do tning, viec can thiet la phai lua chpn dugc chung dai dien khang nguyen tir 4 chung PRRSV. Chung nay phai dat it nhat 02 ti^u chi: i) c6 kha nang kich thich sinh dap iing miin dich nhanh, manh nhat; ii) khang the sinh ra boi chung nay phai nhan biit dupe nhieu chiing vi nit PRRSV khac nhau.
04 chiing PRRSV dupe gay bat ho^t boi formalin b cac nong dp: 0,1%, 0,3% v^ 0,5%. Sau 5 ngay bat hogt vk trung hoa boi NajSaOs, vi nit dupe g^y nhiem tra Igi trfin te bao MARC 145 dl danh gid mirc do bat hogt Kit qua thi nghidm dupe trinh bay trong bang 2.
Kit qua thi nghidm cho thay, tl hko dupe gky nhiim dich vi nit dupe xu ly formalin 0,1% va 0,3% c6 biiu hien co tron ho^c bong troc khoi bl m$t dia nuoi c^y. Nhu vay, n6ng dp formalin 0,1 vk 0,3%
khfing gay bat hoat h o ^ toan doi vdi cac chiing PRRSV.
O cac 16 vi nit dupe xir ly formalin 0,5% da khong phat hien thay hi^n tupng tl bao bj buy hoai sau 5 ng^y gay nhiim, nhu vky nong dO ndy da b^t hogt hodn toan cdc chung PRRSV.
Bang 2. Kdt q u i d i n h g i i miic dO bitho^tdia PRRSV dr c i e ndng dO formahn k h i c nhau Chdng VI
rut
PR-HY 01
PRDNOl
PR-HN 01
PR-VP 01 Formalin
00 0.1%
0,3*
0,5)1 0.1*
0,3%
0,5%
0,1%
0,3%
0,5%
0,1%
0,3%
0,5%
Lidu Ray nhidm TC1D„
10*'
1 0 "
10*'
10^*
Dich VI nit gay nhidm
(ul) 10 ul/
gidng lOiiV gidng 10 ul/
gidng 10 uV gidng
CPE
±
± +
±
±
±
±
2. Dinh gii tuong ddng khing nguydn dia tic
chiing PRRSV ttii nghidm 'fr 04 ehiing PRRSV dupe giy bit lio?t bang j
formalin 0,5% vi nhu hda bing diu Montanide ^ ^ rdi tidm Uuydn cho ttid thi nghidm vdi 02 lin nli*¥
d cie ngiy ttiii 7 vi 15. Theo ddi bidn dOng hidu?'* ] khing thi trdn thd bing phin iing EUSA. Kdt qui thi nghidm dupe tiinh biy trong bi^u d61.
66
NONG NGHlfP VA PHAT TRI^N N 6 N G T H 6 N - Kt 1 - THANG 10/2012KHOA HOC C 6 N G N O H I
Ngiy Bidu dd 1. B i & ddng k h i n g th^ trong m i u thd
du9C Udm PRRSV (EUSA ODjsJ So sinh k h i ning tfio khing thd dje hidu eiia ic chiing PRRSV cho thiy chiing PR-HY 01 cd khi ing t^o dip ling nhanh vi mgnh hon cic ehiing edn li. Tuy nhidn, dd xie djnh ehiing vi nit lim dai didn hing nguydn, 04 miu huydt thanh thu dmic tit thd :uoc tiem PRRSV vd hoat duoe tiiue hidn phin iing
;LISA vdi eie ehiing vi nit edn l?i. Kdt q u i thi ghidm dupe trinh biy trong bang 3.
Bing 3. K^t q u i kidm tra tirong ddng k h i n g iguydn cua 04 chiing PR-HY 01, PR-DN 01, PR-HN
01 v i PR-VP 01 (EUSA 0D.sial,2)
biet duoe nhidu ehung vi nit PRRSV khic nhau.
3. Kdt qui tiio khing thd Ihij dOng qua ldng dd tning g i duoc giy midn djch bdi PRRSV
Chiing vi nit gay bdnh tai xanh PR-HY 01 duoc giy bat boat bing formalin 1% rdi tao nhu vdi dau Montanique ISA 70 dd t^io chit giy midn dich cho g i dd 5 hiin tudi. Gi duoc giy midn dich 3 lin, sau khi giy midn djch, hang min, huydt thanh va long dd tning ga dugc thu thip dd xic dinh hidu gii khang thd tao thinh. Kdt qua thi nghidm duoe thd hidn trong dd thi 2.
^^-^jQwngthd Chiing vi r u T ^ ^ ^ PR-HY 01 PR-DN 01 PE-HNOl PR-VP 01
PR-HY 01 + + + +
PR-DN 01
*
+ + +PR-HN 01
+ + + t
PR-VP 01
t + + + C+: P h d n i i n g E L I S A d u o n g t i n h voi 0 D > 1 , 2 ) K i t q u d p h d n t i n g E L I S A t r u e t i l p c h o thay, iiang t h i d d c h i e u c u a c a c c h i i n g PR-HY 0 1 . PR-DN ll, PR-HN 01 v a PR-VP 0 1 c o k h a n d n g g a y p h a n iing heo voi 0 3 c h i i n g c o n lai. K i t q u a n a y b u o c d a u c o tie k i t luan r d n g 0 4 c h i i n g PRRSV t h i n g h i e m t h u p c iing n h o m h u y e t t h a n h .
Tir k i t q u a b hang 3 v a b i i u d o 1, c h u n g PR-HY 1 da d u p e lira c h p n l a m c h u n g d a i d i ^ n k h d n g guy^n vi c h i i n g n a y d a t d u p e 02 t i e u c h i : c 6 k h a a n g kich t h i c h s i n h d d p i i n g m i i n d i c h n h a n h , lanh n h a t va k h d n g t h i s i n h r a b o i PR-HY 0 1 n h ? n
L o g ;
/ / '
f-f
J Huvet Ihajih i Long d o t n i n g
D6 thj 1. Biln thi6n hi|u gid khdng thi trong huylt thanh vd 16ng do tning gd dugc gdy miin dich v6i
PRRSV
Hieu gia khdng the trong huylt thanh va trong long do tning ga tdng nhanh: trong 6 tuan dau theo doi, hieu gid khdng t h i trong huylt thanh va long do tning la tuong duong nhau vd dat gia tri khoang 11 logj vd dat miic toi da sau 11 tudn gay dap img miin dich voi PRRSV PR-HY 01, tuong irng la 12 logg va 14 log2. Sau 14 tuan theo d6i, hieu gid khang the trong long do tning giam xuong bdng voi hieu gia tai tuan thiie.
Cdc mau huylt thanh va long do tning gd thu dupe a cac tudn theo doi dupe sir dung de thuc hien phan ung trung hoa. Kit qua phan iing trung hoa giua huyet thanh/long do trimg gd voi 04 chiing PRRSV ky hi^u PR-HY 01, PR-DN 01, PR-HN 01 va PR-VP 01 dupe ddnh gid trfin t l bao MARC 145. Kit qua thi nghidm dupe trinh bay b bang 4.
Tdn chiing 'R-HYOl 'R-BN 01 'R-HNOl 'R-VPOl
Huvit thanh gd (61og,) b ckc tudn thi nj 0 9 4
+ + + +
fi + + + +
8 + + + +
10 + + + +
12 + + + +
fhifim 14 + + + +
Long do tning gd (61o^
0
-
2 4 + + + +
6 + + + +
;,) CT cac tuan thi n 8
+ + + +
10 + + + +
12 + + + +
ghiem 14 + + + + (-: im tinh; +: duong tinh)
ONG NGHIEP VA PHAT TRIEN N O N G THON - KY I - THANG 10/2012 67
KHOA H p c C 6 N 0 N O H t
Tit kdt qui bing 4 h-dn ta thiy: bit diu tit hiin thu 4 sau khi giy midn dich, khing thd trong huydt thanh vi ldng dd tning g i d i ed thd trung hda cic chiing PRRSV PR-HY 01, PR-DN 01, PR-HN 01 vi PR- VP 01 oTClDs, hrong img li 10«, 10*«, 10«, 1 0 " .
Muc dich cua vide sin xuit khing thd thy ddng khing PRRSV la dd phdng bdnh tai xanh, vi viy khing thd tdn du sau khi tidm li chi tidu quan trong dd xiy dung quy trinh tidm phdng eho ]t^; trong thi nghidm niy di sir dyng lim 2 thing tudi, cd khdi luong 30 kg. Mdi c i tlid dupe tidm 3 ngiy lidn tidp, mdi ngiy 5 ml dieh ldng dd tning d do pha loing 4 log^,. Dinh ky 5 ngiy/lin, liy huydt thanh l^m dd thuc hidn phin img EUSA xie d(nh hidu gii khing thd tdn du. Kdt qua duoc thd hidn trong dd thj 3.
. , ^°t.
Dd th( 3. Bidn thidn hidu gii khing thd khing PRRSV d l(in thi nghidm
Trong 15 ngiy dau tidn sau khi tidm ldng dd tning ga cd khing thd khing PRRSV, hidu gii khing thd trong huydt thanh Ign dat ngudng tdi da, khoing 7 log2. Sau dd, tir ngiy 20-30 gii tri nay edn khoang 6 log2; theo kdt qui d bing 4, hidu gia khing thd niy vin ed kha ning trung hda dugc PRRSV, vi viy, sau khi tidm khing thd thu dpng 1 thing, Itm thi nghidm ed thd van cd k h i ning khdng bi nhidm PRRSV cudng dpc. Tir ngiy thii 3045, hidu gia khing thd giam nhanh va dat dudi ngudng trung hda dupe PRRSV; kdt qui niy l i ca sd khuydn cio eho vide tidm nhic lai khing thd di dat hidu qui phdng bdnh tai xanh tdt nhit l i sau 1 thing.
IV. KET LUAN V t i l t NGHI
- D i xie dinh duijic lidu TCID50 eua 04 ehiing PRRSV phan lap dupe tit Ipn mic bdnh tai xanh 6 Hung Ydn, Ddng Nai, H i Ndi, Vinh Phuc cd ky hidu PRRSV PR-HY 01, PR-DN 01, PR-HN 01 v i PR-VP 01 hiong ling l i 10<'', 10*-', 10«'», 1 0 " .
- D i lya chpn du^rc chiing d i i didn khing nguyi l i PR - HY 01. Chung vl nit niy bj bit hoat Ij formalin 0,5%.
- Khing thd trong ldng dd tning g i dupe giy mii djch bdi ehiing PR-HY 01 cd k h i ning trung hda PRK cudng dde ttr tuin thli 4.
- Khing thd trung hda tdn luu 1 thing trong Ign, viy djnh ky hing thing tidm nhic l^i khing thd t^
ddng dd ed hidu q u i phdng bdnh tdt n h i t Dd nghi thue hidn cic budc dinh gii hdn didn 1^1 dd t^o CO sd eho vide sin xuit khing thd thg d^n phdng ngira bdnh tai xanh trdn Ipn.
Til lift nUMKHlMl
1. Collins J. E., Benfield D. A , Christianson W, T., Harris L , Hennings J. C , Shaw D. P., Goyal S, M., McCuUough S., Morrison R. B., Joo H. S., Gorcyea D. E., Chladek D. W.,(1992) Isolahon of swine infertility and resphatory syndrome vims (isolate ATCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet Diagn. Invest 1992; 4:117- 126.
2. Akita E. M., Nakai S., (1993) Comparison of four purification methods for the production of immunoglobulin from eggs laid by hens immunized with an enterotoxigenic E. coli shiiin. J. Inuniiiiol Methods, 1993,160, 207-262.
3. Bizhanov G., Vyshniauskis G., (2000) A comparison of three methods for extracting IgY from the egg yolk of hens immunized with Sendai vfrus. Vet Res. Commun., 2000, 24,103-113.
4. BoUen L S., Crowley G., Stodulski G., Hau, J., (1995) Antibody production in rabbits and chickens immunized with human IgG. A comparison of litre and avidity development in rabbit serum, chicken serum and egg yolk using three different adjuvants. J. Immunol. Methods, 1995,191,113-120.
5. BoUen L S., Hau J., (1996) Chicken eggs in polyclonal antibody production. Scand. J. Lab. Anim.
So., 1996,23,85-91.
6. Poison A , Coetzer T., Kruger J., * Maltzahn E., Van der Merve K. ]., (19®
Improvements in the isolation of IgY from the yo»
68 NONG NGHIEP VA PHAT TRIEN NONG THdN - Kt 1 • THANG 10|^),
KHOA HQC C 6 H G NGHl
of eggs laid by immunized hens. Immunol. Invest, 1985,14,323-327.
DEVELOPMENT PASSIVE ANTIBODY AGAINST PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS
Vu Tien Lam, Pham Cong Hoat, Pham Thi Tam Summary
04 strains of PRRSV were isolated from pigs infected porcine reproductive and respiratory syndrome virus in provinces as Hung Yen, Dong Nai, Ha Noi, Vinh Phuc is named PR-HY 01, PR - DN 01, PR-HN 01 and PR-VP 01. Their TCIDso respectively is 10", 10*', 10". 10". SU-ain PR-HY 01 has been selected as a similary antigenic. Concenti-ation of formalin 0.5% could inactivated PRRSV PR-HY 01. Passive antibody in egg yolk belong to hen were Immunized PRRSV PR-HY 01 reach highest at week 11"' but neuUalization antibody appeared at week 4*. Neutralization antibody has been maintained one month in experiment pigs that recommended passive antibody should be injected monthly for prevention porcine reproductive and respiratory syndrome.
Key words: Egg yolk, passive antibody, PRRSV.
Ngudi phin bidn: PGS.TS. Trin Tidn DOng Ngiy nhin bii: 12/8/2012
Ngiy thdng qua phin bidn: 26/9/2012 Ngiy duydt dSng: 3/10/2012
^ O N G NGHlfP VA PHAT TRIEN NONG T H 6 N - KY 1 - THANG 10/2012
