PHAN UfNG TRUNG HOA VIRUT PRRS TREN NUOI CAY TE BAO VA UTNG DUNG
Trdn Thf Thanh Hd', Ken Inui', Phgm Thf Nga', D^ngXudn Sinh', Trinh Quang Dgi', Truffng Anh Dire' vd Nguyin Viit Khong'
T6M TAT
Trung hda virut vdi huyet thanh tren nudi ciy tl bdo cho ph^p ddnh gid dupe mirc huyet thanh Ign mien djch trung hda dugc virut PRRS cd nguin goc khdc nhau, so bd ddnh gid khd ndng mien djch djch the bdo hd.
Trong nghien ciiu ndy ehiing tdi dd thiet ldp phuang phdp trung hda virut (VNT) PRRS tren te bdo Marc 145, bude ddu sir dyng ddnh gid hifu gid trung hda cua Ipn ldnh bfnh trifu chumg tgi thyc dja cd so sanh vdi phuong phdp chin dodn ELISA vd IPMA. Khdc vdi kit qud ELISA vd IPMA, VNT cho thiy huylt thanh Ipn nhifm PRRS chiing Vift Nam chi trung hda virut phdn ldp npi dja, khdng trung hda cac virut cd nguon gdc chdu Au vd Bde My. VNT td ra Id phuang phdp ehilm uu thl hon ELISA vd IPMA trong danh gia dap img mien djch ciia Ign khdng chung dja phuong.
Tir khoa: Virut PRRS, Trung hda virut, Tf bdo Marc 145, Huylt thanh.
Virus neutralization test in cell culture for the PRRS and application
Tran Thl Thanh Ha, Ken Inui, Ph^m Thl Nga, Dang Xuan Sinh, Trinh Quang Dal, Truong Anh Due and Nguyen Viet Khong
SUMMARY
The virus neutralization test (VNT) in cell culture allows the quantitation of antibodies in pig senjm that neutralize the PRRS viruses from different origins, preliminarily evaluates the protective immunity.
In this study we established the method of PRRS virus neutralization in Marc145 cells, in the first steps evaluated the antibody titers in apparently normal pigs comparing the results with those using ELISA and IPMA. Different from the ELISA and IPMA results, the VN ones were obtained as positive only when the Vietnamese PRRS viruses were used but not with the America or Europe origin viruses.
As a conclusion, the VNT could be used to measure the immune answer of pigs towards the local viruses.
Key words: Virus neutralization test, PRRS, Marc145 cells. Serum.
L DAT VAN D £ tai My udc tinh thift hai do PRRS gay ra d mure 720 trifu dd la My. CJ Vift Nam, thift hgi kinh tl Hdi chiing rdi loan sinh sdn vd hd hap d Ign ^^ p ^ g ^.^ ^^ ^j^^^ ^^^^ ^^^ ^.^^^ ^^^ ^^..^
(Porcine Reproductive and Resiratory Syndrome ^^^^ ^^^ ^^^ ^^ ^.^ ^ ^ ^ j ^ ^ ^ ^^^ ^^^. , ^ PRRS) gdy thift hai ldn ddi vdi ngdnh chdn ^, ^^. ^..^ ^^. ^^ ^.j ^^^^ ^xj ,^-^ ^^ ^^ ^.
nudi Ign d Viet nam vd tren the gidi. Hang ndm, ^^^^^^ g . ^ ^^^^ ^.^ ^,^ ^ ^ ^ ^,^ ^^^^^ ^^^^
hilm trong danh myc cdc bfnh ciia Td chiic thii y
^'^"*"y' thl gidi (OIE).
I
i Chuyen gia FAO tai Vi0t Nam.
KHOA HQC KY THUAT THU Y TAP XIX S6 1 - 2012 Cho den nay, Ign Id ddng v^t duy nhit mdc
hpi chimg roi logn sinh sdn vd hd hip (PRRS) [7, 8, 9, 11]. Vifc khing chl djch bfnh dang gdp nhilu khd khdn do chua cd logi vacxin ndo cd thl bdo hp ddn Ign hoan todn phdng ching dugc h^i chirng roi logn sinh san vd hd hip. Dl khdng djnh CO hay khdng khdng the trung hda sau khi tifm phong vacxin hodc sau khi con vdt nhilm bfnh, chiing tdi sir dyng phuang phdp trung hda huylt thanh. Phuang phdp ndy dugc Morrison va cs.
md td ndm 1992, so vdi phuang phdp IPMA khang the. phuang phdp trung hod khdng thl dli vdi virut PRRS Id phuang phdp tifu chuin dimg dl phdt hifn sy xuat hifn khdng thl ch§m.
Phuang phdp ndy dugc Yoon vd cs. cdi tien ndm 1994 bdng cdch pha lodng virut trong huylt thanh thudng, lam tdng dd nhgy ciia phdn img, cho phep phat hifn khdng thl trung hod sdm khodng 11 ngay sau khi gay nhiem; dong thdi, cd thl thay sy khdc bift khi sir dyng cdc chiing virut cd dp tuomg ddng khdc nhau. Sy khdc nhau nay khdng quan sdt dugc khi dimg phuong phdp I FAT. Phuong phap trung hda virut thudng chi ap dung dugc d nhOmg phdng thi nghifm cd dii dieu kifn nudi cay te bdo, it dugc sir dyng thudng quy trong chan dodn. Tuy nhien, phuang phdp nay cd the diing cho nhihig virut PRRS phan l§p dugc cd tinh khang nguyen khdc nhau, dong thdi la phuong phdp tdi uu xdc djnh dg ddi mien djch cua CO thl Ign ddi vdi virut PRRS [7]. 6 Vift Nam, do dieu kifn co sd vgt chit phdng thi nghifm chua cho phfp thyc hifn vifc cdng cudng dpe, vi thl nd cd thl dugc thay thl bdng phuang phdp trung hda virut tren tl bdo.
IL N O I D U N G , NGUYfiN LI$U VA PHUONG PHAPNGHlfeNCtru
2.1. Npi dung
Nudi cay te bdo Marc 145.
- Chuin dd virut PRRS.
Thilt lap phuang phdp trung hda virut PRRS tren mdi trudng tl bdo Marc 145.
Ap dyng phuang phdp trung hda virut dli vdi mpt sd mau tir thyc dja.
2.2. Nguy6n lifu
Tl bdo Marc 145 cd nguin glc tir Uc.
Mdi trudng MEM, huylt thanh bdo thai be ciia hdng Gibco.
3 chang virut PRRS: 0196 (chung phdn I$p tgi thyc dja); chiing chdu Au phan ldp lgi tir vacxin Amervac do Bd mdn hda sinh - mien djch
bfnh ly, Vifn thu y vd chiing Bde My (chiing MLV) do chuyen gia FAO cung cip.
- Chai nudi ciy, dia nudi ciy cua hdng Coming.
Huylt thanh dm chuin vd duang chuin do chuyfn gia FAO cung cip, mdy Ii tdm, tii Ignh, safety cabinate vd mdy mdc khdc trong phdng thi nghifm.
2.3. Phmnig phdp nghifn curu 2.3.1. Nuoi cdy ti bdo Marcl45
Phuong phdp nudi ciy tl bdo dugc thyc hifn theo quy trinh cua CSIRO, Uc (The Commonwealth Scientific and Industrial Research Organisation).
2.3.2. Gay nhiim virut PRRS tren ti bao Marcl4S (dugc thvrc hifn theo hudng dan cua OIE trong chuang 2.08.07)
2.3.3. Xdc dinh sif cd m^ cua virut PRRS bdng phdn urng RT-PCR
Day Id phuang phdp thudng quy tgi Bp mon hda sinh miln djch bfnh ly, Vifn thii y, phan img dugc tiln hdnh theo cdc bude nhu sau:
(1) Tdch ehilt RNA bdng phuong phdp Trizol.
(2) Ting hgp cDNA bdng phuong phdp sur dyng Random primer.
(3) PCR su dyng cdp moi ddc hifu dugc thilt kl dl phdt hifn virut PRRS.
2.3.4. Chudn dg virut
Phuang phdp ndy dugc thvrc hifn theo quy brinh ciia OIE vd tinh TCID50 theo phuang phdp Read «& Muench [I].
Am tinh: Nlu d dp pha lodng dd tl bdo phat triln binh thudng, khdng cd bfnh tich tl bao (gilng doi chumg tl bdo).
Duong tinh: NIU d dp pha loang dd xuit hifn benh tich tren tl bdo (gidng ddi chiing virut).
.A. ,^
2.3.4. Thiet Igp phuang phdp trung hda virut tren moi tru&ng te bdo (theo hudng din ciia OIE trong chuong 2.08.07 vd Virology methods manual)[l,2, 3,7, 8].
m. Kh QUA NGHlfeN CtfU 3.1. Ket qua nudi cay te bdo
De cd te bdo d trgng thdi dang phdt triln va phu day 100% sau 1, 2 ngdy thi lugng tl bdo khi cay chuyen cdn 5x10^ tl bdo/ml, 2x10^ tl bdo/ml.
Vdi mat dp va tinh trgng phdt triln cda tl bdo nhu tren, cdc thi nghifm vf gdy nhiem virut, nhan gidng virut, chudn dp vd phdn ling trung hda diu dat ket qud tdt.
3.2. Ket qua gay nhiem virut PRRS tren te bdo Marcl45
Qua gdy nhiem virut PRRS tren te bdo Marc 145 cho thdy tuy timg chiing ma thdi gian xuat hifn benh tich te bdo khdc nhau vd bieu hifn bfnh bien ciia te bdo cung khdc nhau. Bfnh tich dien hinh la te bdo co cym hinh chiim nho hodc te bao bj phd hiiy hodn todn.
3.3. Xac djnh su cd mat cua virut PRRS bdng phan ihig RT-PCR
Sau klii nhan gidng 3 chiing virut se sur dyng trong nghien ciiu, dl khdng djnh lai sy cd mdt ciia cac chiing virut nay, chiing tdi da sii dyng phuang phdp RT-PCR vdi cap mdi F2R2 cd trinh tu:
Fwd: 5'ATGTTGGGGAAATGCTTGACC3' vaRev: 5'GTTCCGCTGAAACTCTGGTTA3'
Kit qud cho thiy cd 3 chiing virut PRRS sau khi dugc nhan len Uen tl bdo Marc 145 dfu cho kit qud PCR duong tinh. Chiing td vifc gay nhiem virut PRRS vd nhan gidng virut thdnh cdng.
3.4. Ket qua chuin dp cac chung virut PRRS Trong nghien ciiu nay chiing tdi tiln hdnh chuin dp 3 chung virut: (1) chung 0196, (2)
chiing chdu Au; (3) chiing MLV. Mli chiing virut PRRS dugc thyc hifn chuin dO l$p lgi 3 lin, kit qud cdc lin thyc hifn diu gilng nhau.
Bdng 1. Kit qud chuin dd cdc chung virut PRRS TT
1 2 3
Chung virut PRRS 0196 Chdu Au
MLV
TCIDso lO^'^/ml 10®'^/ml 10^'^/ml
3.5. Ket qud thiet lap phdn ung trung hda virut PRRS tren mdi trudng te bdo Marcl45
De thiet ldp phuang phdp trung hda tren mdi trudng tl bdo Marc 145 doi vdi virut PRRS, chung tdi da sir dyng chung virut cudng dfc phdn Igp tgi thyc dja vd mau huylt thanh duong vd am doi vdi phuong phdp ELISA vd IPMA.
Mdu huyet thanh duong Id mdu huylt thanh thu dugc tir Ign dugc gay nhilm virut PRRS nhan tgo vd mdu huyet thanh am la mdu huyet thanh thu thgp tir Ign ciia nhiing viing khdng bj nhilm bfnh PRRS, da dugc sdng lpe bdng phdn ling ELISA vd IPMA.
Phdn ling trung hda virut PRRS tren mdi trudng tl bdo Marc 145 dugc thilt lap tren co sd phdn ling trung hda virut theo hudng dan ciia Virology methods manual [1].
Trong qud trinh thiet lap phuong phdp trung hda virut PRRS tgi Bp mdn hda sinh - mien djch - bfnh ly, thi nghifm dd dugc thyc hifn ldp lgi nhieu Ian, vd sau mdi lin thi nghifm, cdc difu kifn nhu ndng dp CO2, thdi gian li virut vd huyet thanh, nhift df thich hgp cho vifc trung hda virut, v.v. deu dugc toi uu hda sau timg thi nghifm.
Ket qud: Phuang phdp trung hda dd dugc thiet lap gom cdc bude nhu sau:
Bude 1: Chuin bjtl bdo Bude 2: Chuin bj virut
Bude 3: Trung hda huylt thanh Bude 4: Danh gia kit qud
KHOA HQC KY THUAT THU Y TAP XIX S6 1 2012 Am tinh: NIU d dp pha lodng huylt thanh dd
cd bfnh bien tl bdo (huylt thanh khdng trung hda dugc virut).
Duong tfnh: NIU tgi gieng dd ldp tl bdo vin phdt trien tot, bdm ddy vd khdng cd bfnh tfch te bdo.
Doi vdi cdc miu thl nghifm: 6 cdc gilng cd ndng dp huyet thanh cao: Ldp te bdo mpe diu, mjn vd phdt triln tit, khdng cd tl bdo chit, khdng cd bfnh bien tl bdo. d cac gilng cd ning df huyet thanh thip, xuit hifn cdc ddm tl bdo cym lai. sau 5 ngdy cdc ddin te bdo bj bong ra vd chit, klii nhupm khdng cdn hodc rit it tl bdo bdm ddy.
Ket qud cho thiy phdn urng trung hda dd dugc ihiet Igp tren miu huyet thanh duong tfnh vd dm tinh chuan (theo phuang phdp ELISA vd IPMA)
\ ai chung virut thyc dja cudng dpe.
3.6. Kit qud dp dyng phdn ihig trung hda dli vdi mft s6 miu huyet thanh dirong, dm tfnh dli vdi ELISA
Sau khi thilt Ifp dugc phuang phdp, dl tdi dd thyc hifn phdn urng trung hda doi vdi 20 mlu huylt thanh cua Ign sau khi bj bfnh PRRS tgi Qudng Nam duang tfnh vdi ELISA vd 5 miu huylt thanh am tfnh vdi ELISA. Kit qud bude diu cho thiy dli vdi cdc mau huylt thanh dm tfnh ELISA thl ding thdi cho kit qud dm tinh doi vdi phdn urng trung hda. Cdn cdc miu duong tfnh ELISA cao cung cho hifu gid dli vdi VNT.
Chung tdi cung dd sOr dyng cdc mlu huyft thanh tren dli vdi 2 chung virut chdu Au vd Edc My, nhung kit qud deu dm tfnh dli vdi tit cd cdc mau duang tfnh ELISA.
Bang 2. Kit qud thi/c hifn phdn u>ng VNT ddi vdl mdu huylt thanh tir t h ^ dja
TT 1 2 3 4 5 6 7 8 9 10 11 12 13
Ky hifu m § u
243 257 248
246
f
273 269 252 253 275 256 278 259 7
Gia sue
Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn
Lgn ndi
Tuli (thdng)
5 4 4 5 5 5 4 4 5 4 4 4 36
Kit qud ELISA S/P
3,96 3,86 3,78 3.70 3.67 2,99 2,97 2,94 2.92 2,80 2,35 2,28 2,15
Kit qud V N T (log2, Chung V N )
8,00 3.00 7,00 7.00 7.00 7,00 6,00 5.00 6.00 6,00 4,00 4.00 4,00
Kit qud V N T (log2. Chung E U )
0.00 0.00 0.00 0.00 0.00 2.00 0.00 0.00
1.00 0,00 2,00 0.00 0,00
Kit qua VNT (log2, chijng NA)
0.00 0.00 1.00 0.00 0.00 0.00 0.00 0,00 0,00 0,00 1,00 0,00 0.00
TT 14 15 16 17 18 19 20 21 22 23 24 25
Ky hifu mau
5 242 245 260 261 224 222 211 212 214 215 216
Gia sue Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn
Tudi (thdng)
4,5 5 4 4 4 3,5 3.5 5 5 5 5 5
Kit qua ELISA S/P
1,99 1.97 1,33 0,97 0,65 0,50 0,45 0.00 0,02 0,01s 0,03 0,04
Kit qua VNT (log2, Chung VN)
5,00 2,00 6,00 3.00 5,00 3,00 3,00 1,00 1,00 1,00 1,00 1,00
Kit qud VNT (log2, Chung EU)
0,00 0,00 0,00 0.00 1,00 0.00 0,00 0,00 0,00 0,00 0,00 0,00
Kit qud VNT (log2, Chung NA)
0,00 0,00 0,00 0,00 1,00 0,00 0,00 0,00 0.00 0,00 0,00 0,00
Trong nghien ciiu ndy, de so sdnh kit qud ciia 2 phuang p h ^ , chiing tdi sir dyng cdng thiic tinh hf sd tuomg quan:
r = • (=1 1=1 V ' A '
f n \
\ I J
Y,{^\-A Z(>'i~^)
-\2(n-\)s^Sy
i=\ /=!
Bang 3. Hf sd tuo^ng quan giua kit qua ELISA vd kit qud phdn ung trung hda Chung virut
Chung thuc dja Chung Ble My Chung chau Au
Hf s l tuong quan 0,883102 0,276098 0,143956
Qua bang tren cho thiy, cd sy tuomg quan ELISA vdi kit qud VNT ciia cdc chiing chau Au giiia kit qua ELISA vdi kit qud VNT ciia chiing vd Bde My.
thuc dia va khdng cd sir tuomg quan giiia kit qud
KHOA HQC KY THUAT THU Y TAP XIX S6 1-2012 3.7. Ket qud thyc hifn phmmg phdp IPMA dli
vdi mau huylt thanh thyc dja
Trong nghien ciru ndy chiing tdi cung ding thdi thyc hifn phdn irng IPMA dli vdi cdc mlu huylt thanh tir thyc dja. Phdn img IPMA cd df
nhgy vd dO ddc hifu cao, phuang p h ^ ndy thudng chl dugc sir dyng trong cdc phdng thi nghifm cd dilu kifn nudi ciy te bdo vd gay nhilm virut.
I T
1 2 3 4 5 6 7 10 12 14 15 21 22 23 24 25
Bdng 4. Kit qud thyc hifn phdn
Ky hifu mdu
243 257 248 246 273 269 252 256 259 5 242 211 212 214 215 216
Gia sue Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn Lgn
Tudi (thdng) 5 4 4 5 5 5 4 4 4 4,5
5 5 5 5 5 5
u>ng IPMA ddl vdl miu huylt thanh thi/c dja Kit qud
ELISA S/P ratio 3.96 3,86 3.78 3,70 3,67 2.99 2,97 2,80 2,28 1,99 1.97 0.00 0,02 0,01 0,03 0.04
Kit qud IPMA (ChCing VN)
4,00 4.00 4,00 4,00 4.00 4.00 4,00 3,00 3,00 4,00 3,00 0.00 0,00 0,00 0,00 0.00
Kit qud IPMA (chung EU)
3,00 3,00 3,00 3,00 3.00 3.00 3.00 3.00 2.00 2.00 2,00 0.00 0.00 0.00 0.00 0.00
Kit qud IPMA (chung NA)
3.00 2.00 3.00 3.00 3.00 4,00 3.00 2.00 2,00 3.00 2,00 0,00 0.00 0.00 0.00 0.00
Ket qua bude dau cho thiy khi thyc hifn dli vdi cd 3 chiing virut diu cho kit qud dm tinh vd duong tinh rd.
Bang 5. Hf sd tu-ang quan giCra kit qud ELISA vd kit qud phdn img IPMA
Chung virut Chung thyc dja Chung Blic My Chung chdu Au
Hf sd tuong quan 0,951706 0.894025 0,977332
Qua bdng kit qud ve hf s6 tuomg quan cho thay kit qud thyc hifn phdn img IPMA tren cd 3
chdng diu cd tuang quan dli vdi kit qua cu phdn img ELISA.
cua
Bdng 6. Hf sd tuang quan giOa kit qud trung hda vd kit qud phdn irng IPMA Chung virut
Chung thyc dja Chung Bic My Chung chau Au
Chung thyc dja 0,866012 0,210092 0,210092
Chung Bic My 0,210092 0,213021 0,402374
Chung chdu Au 0,210092 0,402374 0,220564
Chi cd sy tuong quan gi&a ket qud cua phdn ling trung hda vd IPMA cua chiing virut thyc dja.
Con ket qua cua cac chung khac khdng cd sy tucmg quan.
IV. Kh LUAN
Trong nghien ciiu nay chiing tdi cd nhOmg ket ludn nhu sau:
Phan ling trung hda te bdo ddi vdi virut PRRS da dugc thiet lap tai Bd mdn hda sinh mien dich - bfnh ly, Vifn thii y.
Phuang phdp dugc thiet lap gdm cac cdng dogn: (a) Chuan bj te bdo; (b) Chuan bj virut;
(c) Trung hda huylt thanh; (d) Dgc ket qud phan ling.
Bude ddu dp dyng phuang phap trung hda ddi vdi mdu thuc dia vd cd su so sdnh ket qua vdi 2 phuang phdp ELISA vd IPMA.
Ket qud trung hda ciia cac mau huyet thanh cua gia sue bj bfnh tir thuc dia deu am tfnh ddi vdi cac chung chau Au va Bde My.
- Ddi vdi chung virut chau Au vd Bac My, ket qua cua phan ling ELISA khdng tuomg dong vdi ket qua ciia phdn ling trung hda virut.
TAI LifiU THAM KHAO
1. Brian WJ Mahy and Miliar O Kangro (1996).
Virology methods manual, Chapter 6, p 108; p 195-199.
2. Enuh Rahario JUSA, yuji IN ABA, Michihiro KOUNO, Omasu HIRUE, Hisao YASUHARA (2005). Slow reacting and complement requiring neutralizing antibody in swine infected with
PRRS vims. Journal of General Virology, 86, p 1943-1951,
3. E, Albina (1998). Immune responses in pigs infected with PRRS. Veterinary Immunology and Immuno-pathology, p 49-66.
4. I. Di'az,l,2 L. Darwich,l,2 G. Pappaten-a,3 J.
Pujols 1,3 and E. Mateul,2. Immune responses of pigs after experimental infection with an European strain of Porcine reproductive and respiratory syndrome virus. Journal of General
Virology (2005), 86, p 1943-1951.
5. Gonnie Nodelijk (1996). Comparison of commercial ELISA and immune peroxidase monolayer assay to detect antibodies directed against PRRS. Veterinary Microbiology, p 285- 295.
6. Michael O'Connor Irish (2002). Detection of antibody to PRRS virus by ELISA and IPMA,
Veterinary Journal, p 73-75 .
7. Nettebotner (1997). Diagnosis of PRRS.
Veterinary Microbiology, p 295-301.
8. OIE Terrestrial Manual (2008). Porcine reproductive and respiratory syndrome. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, p 1116-1127.
9. J.Plana-Duran (1997). Efficacy of inactivated vaccine for prevention of reproductive failure induced by PRRS. Veterinary Microbiology, p 361-370.
10. R.W.Wills (1997). PRRS a persistent infectioH.pdf Veterinary Microbiology, p 231-240.
11. Zygmunt Pejsak (1997). Clinical signs and economic losses caused by porcine reproductive and resiratory syndrome virus in a large breeding farm. Veterinary Microbiology, p 317-322.
