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KHAO SAT S U LUU HAIMH VA PHAIM T I C H GIEM O R F S
CLIA PRRSV PHAI\I LAP TAI KHAIVH HOA
^ VQ K h k Hiing^, D6 Vdn 1h^, Trinh Thu Hing^, Ld Thdng^, ( J? t/ / 3 ^ NguySn Tlii Thu Giang^, Ddo Hodi TTiuS Ld Tlii Nhu6n|^
T6MTAT
Tu 100 mdu b ^ h pham thu thdP tir cdc d dinh va Ion nghi mdc b^nh tai xanh thu thap tai Khanh Hoa tit 2011-2014 da phdn lap dugc 75 mdu vi nit tai xanh. Moi vi nit dupe phdn Idp tii mot benh pham (tu mot ca thi Ion). Trinh tu nucleotit cua do^n gien 0RF5 tii 75 vi nit da duoc phan tich h ^ g ky thudt phan nhdm cCia Cong ^ MJ-Biologics, Hoa Kj- (MJPRRSV Grouping Technology); k^t qua cho thay cdc chiing ndy d^u thuoc nhom Dl- Trung Qu6c. Kit qua kiem ba TC1D50 cua 25 chting PRRSV dao dong tii 4.5 Ig den 6,2 Ig TCID50/ml. Da ebon 3 chiing NT02, NH02 va VNOl gay nhiSm vdi lieu lO^TCIDSO p-en 15 Ion (mdi chung gay nhiim cho 5 Ion); ket qua cho th^y sau gSy nhidm Ipn hieu hi&n cdc tneu chiing lam sang nhu sot cao
>41'*C, cd not do tia, tai cd not mdu dd xanh tit ngay thu 5-7.
Tukhda: Bdnh tai xanh, MIPRRSVgrouping technology. ORFS, PRRSV. TCID50.
Hdi chiing rdi loan sinh sdn va hd hapd Ign (PRRS- Porcine reproductive and respb-atory syndrome), cdn gpi Id bdnh "tai xanh" (Blue Ear), la benh truyen nhiem cElp tinh nguy hidm, do vi nit PRRS (PRRSV) thudc bd Nidovirales, ho Arterivirales, chi Arterivirus gdy ra. PRi^ lan dau tidn dugc phat hien b My vao ndm 1987, tai chau Au (Diic nam 1990, Ha Lan nam 1986) vd tai chau A vdo ddu ndm 1990 (Wensvoort et al., 1993). PRRSV dugc chia ra 2 ladu gien chinh la kidu gien cd ngudn ^6c bi chau Au (EU) cd tdn gpi Id Lelystad vd kidu gien cd ngudn gdc bi Bdc My ( N ^ cd tdn goi Id VR2332.
Su gidng nhau ve cau tnic chudi nucleotit cua vi nit thudc hai chung trdn vao khodng 60% (Metwally et al., 2010). Trong nhiing nam gan day, cdc 1^ thudt sinh hgc phan tii duoc su dung dd phdn tich mdi quan he phat sinh loai vd nhiing bien ddi he gien cua cdc chung PRRSV dugc phan lap d nhiing dia didm khac nhau.
He gien cua vi nit PRRS Id phdn bi ARN sgi don duong, cau hiic hrong hi cdc khung dgc md (open reading frame, ORF) ciia Coronavirus, gom 7 khung dgc md goi Idn nhau, ma hda cho 7 protein ciia vi nit gdm: GPI, GP2, GP3, GP4, GPS, M va N. Trong so dd, protein GPS (glycoprotein 5) dugc ma hoa boi
' PhSn vien Thii y mign Tmng
^ Chi cue Thii y Khanh Hda
' Hoc vien cao hpc Trudng Dai hoc Nha Trang
0RF5, la mdt protein dugc glicozil hod, gan vdi mang hgc ngodi ciia vi nit PRRS, cd tinh khdng nguyen manh vd chiu trdch nhidm gdy benh cua vi nit (Vu Khac Himg vd cs, 2015). Nhung nghien ciiu vd b i ^ ddi hd gien ciia cdc chung PRRSV chu yeu trdn gien ORFS md hod protein GPS (glycoprotein 5), Id gien thudng xay ra hidn tugng dot bidn diem cua mot sd chiing vi nit khi phdn tich trinh tu gien dich (Ansari et al., 2006; Andrea et al., 2014).
Vl nit PRRS xuat hidn tai Viet Nam ndm 1996 v^
nhanh chdng Idy lan trong dan Ign ca nude. Su xuat hidn cua chung vi nit PRRS mdi 1dm giam hidu luc cua cdc vdc xin dang dugc sii dung. Theo sd lieu cua Chi cue Thu y bi thdng 9/2007 tai Khdnh Hda da xay ra 04 dot dich vdo nam 2007, ndm 2010, nam 2011 vd nam 2012 vdi tdng sd Ign mdc bdnh lac benh 1^
20.822 con. s6 con chet Id 975 con, bude tidu hiiy 61.013 Ign, udc tinh thidt hai do bdnh tai xanh gay ra Id 42.996 trieu ddng. Tuy nhien, chua cd nghidn cuu nao vd vide xdc dinh dac tinh dt truyen cua cdc chiing PRRSV h-dn dia bdn Khdnh Hda. Dd cd co sd khoa hpc cho vide lua chgn loai vac xin phdng benh PRRS phu hop rieng cho Ign nudi tai Khdnh Hda chiing tdi tien hanh "Khio sat su luu hinh va phin tich gien 0RF5 cua PRRSV phin lip tai Khinh Hda".
2 . V A I UEU VA PHUONG PHAP NGHIDN CUU 2.1. Vdt lieu
- Mdu bdnh phdm (phdi, hach bach huy^t, Id Idch, huyet thanh) dugc fliu tai cdc d dich tai xanh trdn tinh Khdnh Hda bi nam 2011 ddn 2014.
7 8 ^
KHOA HOC CdNG N G H l
Bd kit tdch chidt QIAamp Viral Mmi Kit (Qiagen), bd kit One step RT-PCRkit (invitrogen).
- Cdc loai sinh phIm dung cho phdn ling PCR.
Mdi trudng nudi cay: mdi trudng EMEM, td bao MARC145, huydt thanh bao thai be do Hang Gibco cung cdp.
- Lgn con 6 tudn tudi khdng cd khdng the khdng VI nit tai xanh (xdc dinh bang phuong phdp E l i S ^ khdng mang vi nit tai xanh (kdt qua RT-PCR dm tinh).
2.2. Phuong phdp nghidn cihi
Phuong phap phan lap vi nit theo hudng ddn cua OIE trong chuong 2.08.07- OIE Terrestrial Manual 2010.
ARN dugc tdch chiet ttr mdu bdnh pham bang each su dung bd kit QIAamp Viral Mini Kit- QIAGENE, tiheo hudng ddn cua nhd sdn xudt
Khudch dai doan gien 0RF5 cua vi nit PRRS bdng phuong phdp RT-PCR su dung bd kit Invitrogen Supersrip III One - Step RT-PCR.
M6i
Go3F (Forward)
Go5R (Reverse)
Trinh tii moi
GAGACCATGAGGTGGGCAA CGGCAAAAGCACCmTGT
Kich thuoc san
pham PCR
721 bp
- Tdch chiet vd tmh sach san pham RT-PCR tir gel bdng bd kit sinh pham QIAquick Gel Extraction Kit San phdm RT-PCR dugc gui den gidi trinh tu tai Cdng ty Macrogen Hdn Qudc.
Phdn tich trinh tu gien theo ky thuat phan nhdm cua Cdng ty MJ. Biologies (hinh 1).
Bd tri thi nghiem gdy bdnh thuc nghidm trdn lgn theo md ta cua Td Long Thdnh vd cs, 2008.
Chung phan lap
Chung Bac My
I I M I I I I
D-l 2 3 * 5 6 7 8
I I I I I I I I
S-1 2 3 4 5 6 7 8
Chung cMu Au
3. KET QUA VA THAO LUAN
3.1. K^t qud ki^m tra su cd m3t cua PRRSV trong mku bdnh phSmvd phdn % v i nit
Cac mdu bdnh pham sau Idn dua ve phdng thi nghidm, dugc xu ly vd chidt tdch ARN su dung cho phan ling RT-PCR Ket qud RT-PCR cho thay 75/100 mdu cho k^t qud duong tinh vdi cdp mdi G03F vd
Hinh 1. Sa d6 phdn nhdm vi nit tai xanh
G05R vd san pham cd kich thudc 721 bp. Kdt qua duoc thd hidn d hinh 2.
75 mdu benh phdm cho kdt qua RT-PCR duong tinh, tidn hdnh phdn lap vi nit tai xanh trdn mdi birdng b-dn td bdo MARC-145. Kdt qua da phdn Idp dugc 75 vi nit tai xanh trdn tdng sd 75 mdu benh pham, chiem ty le 100% so vdi kdt qua RT-PCR duong tinh.
N O N G NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 12/2015 79
KHOA HOC CdNG N G H £
Hinh 2. K^t qua ET - PCR phat hi$n vi nit tai xanh cac mdu b0nh ph£m
Chii tiiich: - M: Thanh ABN chuin; P: Miu ddi chdng duong (vinit tai xanh TB-8); N: miu ddi chiing im; 1-18: Cac miu bdnh phim
Tu kdt qud phan tich trinh tu gien ORFS, 25 mdu vi nit tai xanh dugc chgn de xJU: dinh TCID50; k^
qua cho thay TC1D50 cua 25 mdu vi nit dao dong tii 4,5 loglO den 6,2 loglO. Kdt qua ndy phu hpp vdi k^
qua nghidn ciiu cua chiing tdi (Vu Khdc Hung va cs.
2015) va kdt qua nghien ciiu ciia Tran Thi TTianh Ha vd cs. (2012); nhdm tdc gid nay nhan Ihay TCID50/ml cua cdc chung 0196, chdu Au va MLV lan lugt Id 6,5 Ig. 5,5 Ig vd 6,7 Ig TCID50/ml. Mdt nghifin Cliu Midc cua Kamiychuk vd cs. (2010) cho thay hieu gid vi nit ciia cdc chung Belgium A vd Lena phdn lap tir bdnh pham dao ddng tir 2,2 Ig den 7,7 Ig TCIDSO/ml.
Tir kdt qud xdc dinh TCID50 chgn 03 chiing co ket qud TCID50 cao nhat dd Idem tra ddc luc trSn lgn, mdi chiing kidm tra trdn 5 lgn, tidm bap vdi lieu lO^TCID 50 theo Td Long Thdnh vdcs. (2008).
Bang 1. Ba chung vi nit tai xanh diing d^ gdy nhi^m trdn lgn
Hinh 3. Phdn lap vi nit tai xanh NT02 d-en t^ bdo MARC145.
3.2. K^t qua giai brinh tu vd phan tich gien ORFS Sau khi phan lap duoc vi nit trdn te bao MARC145, chiing tdi chiet tach ARN, thuc hidn phan ling RT-PCR, tinh sach san pham PCR vd giai trinh tu gien ORFS cua 75 mdu vi nit phdn IdP trdn. h^
dung ky thuat phdn nhdm cua Cdng ty MJ-Biologics de phan tich trinh tu gien ORFS; ket qud cho thay 75 mdu vi nit phan Idp tai Khdnh Hda ddu thudc phdn nhdm Dl- cd ngudn gdc tir Trung Qudc. Kdt qua ndy phu hgp vdi nghien ciiu trudc ddy, phdn tich trinh tu gien ORFS ciia 300 chung phdn lap tir midn Bac, Trung, Nam cho thdy da s6 (96,15%) cdc chiing vi nit tai xanh dang luu hanh tai Viet Nam thudc phdn nhdm Dl cd ngudn gdc tu Trung Qudc vd chi mdt ty Id rdt nhd (3,85%) thudc bidn the D2, D4, D6 vd S3 (Nguydn Diic Tan vd cs., 2015).
3.3. K^t qua xdc dinh TCID50 vd gdy nhifei trdn lgn
STT 1 2 3
Chung NT02 NH02 VNOl
TCID50/ml 6,11E
61g 6,21g Ket qud cho thay cd ba nhdm lgn dugc tidm vi nit tai xanh vdi lidu lO^TCID 50 d^u co cdc bidu hien lam sdng nhir. sot cao >41''C, cd not dd tia, tai cd not mdu do xanh tu ngdy thii S - 7 sau khi dugc gdy nhidm. Quan sat thay lgn bi bo vd bdt hoL Trieu chiing bidu hien giong nhu md td trudc day cua Wemike vd cs. (2012) khi nhdm tdc gia ndy gay nhidm chiing vi nit tai xanh dOc luc cao vdi lidu 5,8 Ig TCID50/ml. Tir ngay thii 10 nhidt dd gidm dan nhung vdn cdn cd lgn sot Sau do tat cd ddu hdi phuc, \
KHOA HOC CdNG NGH|
khdng CO lgn chet Kdt qud ndy tuong ta nhu ket qua cua Td Long Thanh vd cs. (2008).
Song song vdi quan sdt tridu chiing 1dm sdng hen hdnh Idy mdu todn bd lgn de kidm tra vi nit tai xanh vdo cdc ngdy 7, 14 vd 21 sau khi gdy nhidm.
Bang phuong phdp RT-PCR phdt hidn dugc vi nit tai xanh trong huydt thanh vdo ngay thii 21 sau gdy nhidm, tuy nhidn ndng dd vi nit cdn lai rat thap (gid tri Ct h-ong huyet thanh lgn duoc tidm NT02, NH02 vd VNOl hung binh Id 30,18, 30,81 vd 35,71 theo thii tu).
Trong thi nghidm ndy, chgn ngdu nhidn 2 lgn trong mdi nhdm lgn gdy nhidm de m6 kiem tra benh tich vao ngdy thii 10 sau khi gdy nhidm; k^t qua md kham cho thdy cd benh tich vidm phoi (hinh 4), gan cd d6m trang hoai tu (hinh 5), hach 1dm ba simg to (hinh 6), tim bi simg vd mdng bao tim bi xudt huyet (hinh 7). Ket qua ndy tuong duong vdi kdt qud cua Td Long Thdnh vd cs. 2008, khi tidn hdnh gdy bdnh thuc nghiem trdn lgn vdi lidu hdm bdp 10^
TCIDSO/lgn, khdng cd lgn gdy bgnh thirc nghidm nao chdt, lgn cd bieu hien sdt cao; khi md kham quan sdt thdy benh tich vidm phdi, Idch xudt huyet be mat vd gan cd ddm trdng.
Hinh 4. Hidi dia heo gdy nhidm PRRSV bi vidm
Hinh 5. Phdi bi viem, gan cd ddm trdng ho^i tu
Hinh 6. H^ch 1dm ba sung to 4. m LUAN
Cac mdu vi nit PRRS phdn Idp dang luu hanh tai Khdnh Hda thudc phdn nhdm Dl cd ngudn g6c tir Trung Qudc.
Khi gdy nhidm thuc nghidm 3 chung vi nit tai xanh tren lgn, tat ca lgn thi nghiem ddu cd trieu chimg benh tich di^n hinh cua bdnh tai xanh.
TAI UEU THAM KHAU
1. A n d r e a Ladinig, J a m i e Wilkmson, Carolyn Ashley, Susan E. D e t m e r , Joan K. Lunney, G r a h a m Plastow, a n d J o h n C. S., 2014. H a r d i n g - V a n a t i o n in
Hinh 7. T u n s u n g , m d n g b a o tim x u d t h u y d t Fetal O u t c o m e . Viral Load and ORFS S e q u e n c e M u t a t i o n s in a Large Scale Study of Phenotypic R e s p o n s e s to Late Gestation E x p o s u r e to T y p e 2 Porcine Reproductive and Respiratory Syndrome, PLoSOne9{'i),pp-2^^-
2. Ansari, L H., Kwon B., Oorio F. A and Patbiaik A. K., 2006. Influence of N-linker glycosylation of porcine reproductive and respiratory synddrome vu-us GPS on virus infectivity, antigenicity, and ability to induce neutralizing antibobies. / Virol 80 (8), pp. 3994-4004.
N 6 N G NGHIEP VA PHAT TRIEN NONG THON - KY 2 - THANG 12/2015
KHOA HOC C6NO N G H t
3. Christopher-Hennings J., Nelson E. A, Nelson J. K., Rebecca J., Hines, Sines, Sabrinina L, Swenson, Howa L, Swenson, Howard T, Hill, Jeff J.
Zimmerman, Jon B. Kkatz, Michichael J. Yael J.
Yaeger, Christopher C. L. Chase, and David A Benfield, 1995. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR. / CUn. Microbiol. 33:1730-1734.
4. Egli C, Thur B., liu L, 2001. Quantitative Ta qMan RT-PCR for the detection and differentiation of European and North American strains of porcine reproductive and respiratory syndrome virus. /
Yuvl. Methods 98 63-75.
5. Han J., Wang Y,. Faaberg K. S., 2006.
Complete genome analysis of RFLP 184 isolates of porcine reproductive and respiratory syndrome virus. VmisRes.J. 122(1-2): 175-182.
6. Kumar S., Dudly J. Nei M. and Tamura K, 2008. MEGA: A biologistK;entric software for evolutionary analysis of DNA and protem sequences.
BrieBngs in Bioinlormatics (9): 299-306.
7. Li Y., Wang X., Jiang P., Chen W., Wang X., 2008. Genetic analysis of two porcine reproductive and respiratory syndrome viruses with different virulence isolated in China. Archives of Vu-ology 153,10:1877-1884.
8. Metwally S., Mohamed F., Faaberg K., Burrage T., Prarat M., Moran K, Bracht A, Mayr G., Bermnger M., Koster L, To T. L, Nguyen V. L, Reising M., Landgraf J., Cox L, Lubrothand J.
Carrillo C. (2010). Pathogenicity and Molecular Characterization of Emerging Porcine Reproddctive and Respiratory Syndrome Vu-us in Vietnam in 2007. Transboundary and Emerging Diseases 57:
315-329.
9. OIE Terrestrial Manual, 2010. Chapter 2.8.7.
Version adopted by the World Assembly dl Delegates of the OIE in May 2010 porcine reproductive and respiratory syndrome, 2.6.7.
10. NguySn Diic Tin, VO Khac Hiing, Byoung Kwan Kim, D6 Van KhiSn, DO Van Tin (2015). Phan lap va tuyIn chon mpt so chung vi nit tai xanh (PRRSV) doc luc cao d^ san xuat vacxin phong b^nh cho Ion. KhoahocKythuitThliy.'Maib.2A-'i2.
11. Tran Thi Thanh Ha, Ken hiui, Ph?m Th|
Nga, Dang XuSn Sinh, Trinh Quang D^, Tmong Anh Diic va NguySn Vi& Khdng (2013). Phan ling trung hoa vi nit PRRS tr6n nu6i cay t^ bao va ling dung. Khoa hoc Ky tiiuit Thiiy 1.27-33.
12. To Long Thanh, Nguyen Van Long vd cpng su (2008). K^t qua chan doan vd nghi&n ciiu vi nit gay hdi chiing sinh san va ho hip (PRRS) tr6n lgn i Vitt Nam tir thang 3/2007 dfo 5/2008. T^ c4|
Khoa hoc Ky tiiuit Thu y. 5.5 -16.
13. Vu Khdc Himg, Do Van Tin, Ho Van HiSp, NguySn Thi Thu Hang (2015). D4nh gia su dn dufl cua mpt so chiing vi nit gay h^i chiing rdi loan sinli san va ho hip thugc nhom Dl- Trung Quoc. Khoa hocKyOiuitThliy. 3-2015,15-19.
14. http://www.mjbio.com/index.php PREVALANCE AND ANALYSIS OF ORFS GENE OF PRRSV ISOLATED IN KHANH HOA PROVINCE
Vu Khac Hung, Bo Van Tan, TMnh Thu Hang, Le Thang, Nguyen Thi Thu Giang, Dao Hoai Tliu, Le Tlii Nhuong
Summary
A total of 100 samples collected from the outbreaks and suspected pigs of PRRS (from 2011-2014) in Khanh Hoa province were used for isolaHon of PRRSV sfrains. As a results, 75 PRRSV sttains were isolated and each strain from one pig. Sequence of the gen ORFS from 75 PRRSV sfrains was amplified and analyzed by if usmg MJPRRSV Groupmg Technology of MJ-Biologics company, USA. The analyzed results show that 75 , PRRSV strains belong to Dl- Chinese group. The results of TCID50 testing of 25 Isolation were from 4.2 Ig to 6.3 Ig. 3 PRRSV sfrains to infect 15 6 - week - old pigs were selected (each strain infect 5 pigs). The body temperature of all Infected pigs reached 41"C and have symptoms like red to purple dermal discoloration and purple and blu ear^ at five to seven day post infection.
Keyword: Blue ears disease, MJPRSSVgrouping technology, ORFS, PRRSV, TCW50.
Ngufti phin bi«n: PGS.TS. T6 Long Thinh Ngay nhjn bii: 26/6/2015
Ngiy thdng qua phan bifti: 27/7/2015 Ngay duy$t dang: 3/8/2015
