Tap chi Khoa hpc vfi Phfit triln 2012: Tap 10, sfi 2:301 - 306 TpU'6NG DAI HQC NONG NGHIgP HA NQI
CHAN DOAN HOI CHlKNG ROI LOAN HO HAP VA SINH S A N ( P R R S ) i LON CON CAI SQA BANG KY THUAT BENH LV VA KY THUAT RT - PCR Diagnosis of Porcine Reproductive and Respiratory Syndrome (PRRS) in Weaned
Pigs by RT - PCR and Pathologycal Methods Nguyen Thi Lan, Nguyen Thj Hoa Khoa thd y, Trudng Dgi hgc Ndng nghidp Hd Ngi
Dja chi tac gia lien hp: [email protected] Ngay gCri bai: 6.03.2012 Ngay chap nh^n: 17.04.2012
TOM TAT
Hpi chu-ng rdi lo^n hd hdp vfi sinh san (PRRS) gay thi^t h^i Id-n cho ngu-^i chfin nudi Ip-n. D$c bi^t l^n con sau cai sira cd t^ 1$ mfic b^nh cao vi dfiy Ifi li>a tu6i Ip'n dh mfin cfim, si>c Oh khfing y l u . NghiSn ci>u nay duyc t i l n hfinh tren dfin l^n sau cai sira ti> 3-6 tuin tu6j. Cac Ip'n sau cai sOa bj mfic PRRS c6 bilu hi$n bo fin, chfin fin, s6t cao, da m i n dd, khd thd*. Phdi ipm b^nh cd hifn tipp'ng sung huyit, xult huyet, viem ph6i. H^ch ifim ba svng to, tg mfiu. Th^n c6 x u i t huyit llm t i m . Xoang bao tim tich nvo'c. Cac ton thu^'ng vi t h i bao gom tham nhilm t l bao viem, p h i quan phi viem ho$c viem ph6i thCiy, xult huyit c l u th|in, b l th^n. K i t qufi phan ung RT-PCR da cho thly cfic Ip'n c6 bilu hi^n tri|u chi>ng iam sang, b^nh tich nhir tren deu mfic PRRS.
Tip khoa: PRRS, ip'n con cai sira, RT-PCR.
SUMMARY
PRRS causes severe economic loss for the fanners. Post weaning piglets are highly infected with PRRS because of high sensitivity and low immunity with PRRS. The present research was carried out in post weaning piglets at age from 3 weeks to 6 weeks. The piglets showed deprssion, anorexia, fever, red skin, dyspnea. The piglets also showed congested and hemorrhage lung and pneumonia, and enlarged lymph nodes, hemorrhage kidney. The microscopic lessions consisted of inflammatory infiltration in lung, pneumonia or bronchopneumonia, hemorrhage in pelvis of kidney. The piglets with the above clinical signs and pathological findings were diagnosed to be infected with PRRS by RT-PCR.
Keywords: PRRS, weaned pigs, RT-PCR
1. DAT VAN DE Nidovirales, cd cfiiii tnic he gen la RNA spi dpn dUPng, gdm 7 khung dpc md khac nhau ma hda Hoi chiine rra loan hd hfin va sinh san , - . - \„,.:',„//-< ^u loo-n
° - *^ cho eac protem cua virus (L-avanaugn, 199 /).
(Porcine Reproductive and Respiratory , , o J •nTiT)ci\ 1- J- v u- u 4_ -- i,= PRRS dfic biet nguy hiem d ldn eon sau Syndrome - PRRS) la dich benh truyen nhiem . . & j .
, .J ' 1 V. i_ - •..--^ L - 1 - L ^-' cai siia vi day la mot trong nhiSng nhom iPn nguy hiem cua loai ldn, gay thiet hai kinh te . , -^ • ^ ^ iA_ 1, - L L- ' -• 1- DDDc- lA- .»A' ^kn Cam nhfit vdi virus PRRS. Ldn con sau ldn cho nganh chan nuoi ldn. PRRS lan dau , , .^ , . ^ 7 ' »o (-•- J.,- u-i. i.-- ;.HT~ -' man /K" ff u cai Siia thUOng cd bieu men sot, da mfin do, tien diTdc phat men d My nam 1987 (Keftaber, ,, , , " , . • > , . ,,,,•• ,r., -,non\ -('T- T>T>TIOI5 J 5 ..•- A ..^ u- la mdt moi, bo fin, CO biou hicn khd thd (Chung 1989). Virus PRRS lfin dfiu tien dUdc phan lfip ' ^ ' • ^^ ^ ,.-., a., -i" J-1- 1 TI- T .*..- • ^- I, 1- vfi cs, 1997; Done va CS., 1996).
tij mpt 0 dich d Ha Lan, dUdc xac dinh la ' '
nguyen nhan chinh gay ra hdi chiing rra loan PRRS mdi xuat hi§n b Viet Nam tii vai hd hap vfi sinh san b ldn. Virus thupc nhdm nfim trd l^i day nhUng da gay ra nhiing thiet Arterivims, thuoc hp Arteriviridae, bd hai vo ciing to ldn cho nganh chfin nudi ldn
Ch^n doan hpi chu-ng r6i logn hd h^p va sinh sgn (PRRS) d lyn con ... va kg thugt RT - PCR ndi chung vfi khd khfin trong vi^c ch^n dofin
phat hi^n dich b^nh PRRS ndi rigng. Lpn mfic PRRS thadng k^ phfit cttng nhid'u b$nh nguy hi^m khfic til vi khu^n ciing nhu virus gay b§nh khfic lfim cho cdng tfic ch^n dofin g^p nhid'u khd khfin.
Th^ ky XXI lfi th^ ky cua cong ngh0 sinh hpc cdng vdi 8\f phfit tri^n manh md cua ky thu$t sinh hpc phfin tii da md ra nhflng tri^n vpng nghidn cdu vd ciing to ldn. Ap dyng ky thu^t sinh hpc phan tit dfic bi^t lfi ky thu^t RT- PCR cho phdp ch^n doan nhanh, chfnh xfic cfic b^nh do virus gfiy ra mpt each dfic hi^u trong dd cd h^i chdng rdi loan hd hfi'p vfi sinh san ddng thdi gdp phin xay di^ng chiS'n lifpc ki^m sofit dieh bpnh tren dfin lpn b Vi$t Nam. Nghien cflu nfiy da cho thfi'y eae keft qua nghign ctfu ling dung ky thu^t RT-PCR de ch^n doan PRRS b ldn con cai siia.
2. VAT LIEU VA P H U O N G P H A P 2.1. Vat lieu
Nghien ciiu nfiy diJpc tien hanh trgn cac dfin lpn con cai siia 3-6 tu^n tuo'i nghi mSc PRRS trgn dia bfin mot sd tinh nhii Hang Yen, Bfic Ninh, Hfi Ndi. Cac lpn nghi mic benh dupe quan sat, theo ddi tieu sut, tripu ehiJtng lam sfing, vfi mo kham thu thfip cfic ed quan bgnh ph^m eua ldn nha pho'i, lach, hach lam ba, rupt.
2.2. Phrftfng ph&p n g h i e n cihi - PhilOngph&p quan sat cac bien doi b$nh If: Trac tiep quan sat cac tripu chiing lfim
sfing, m^ khfim, quan sat cfic bid'n doi b$nh IJ d^i th^ cua cfic lpn sau cai s3a. Tidu ban vi the dapc tidn hfinh theo phUdng phap t^m diic paraffin thadng quy cua phdng thi nghidm B§
mdn B^nh 1^ Thu y, Khoa Thu y, tradng D^i hpc Ndng nghidp Hfi N^i. MSu bdnh ph^m cila lpn nha ph^i, Ifich, hach lfim ba, rudt dupc cd dinh trong formol trung tinh 10%. Sau d6, dupe tfi'm due paraffin, dt tigu ban bfing may c^t microtom vfi nhudm tidu ban bfing phaong phfip Hematoxylin-Eosin (HE).
- Phuang phap RT-PCR: bao gdm cac bade tach chig't RNA eua virus vfi ckc bade thi^c hi|n ky thufit RT- PCR. RNA cua virus dapc tach chidt bfing kit QIAamp d6 tien hfinh phan dng RT- PCR. Quy trinh taeh ehiet RNA cua virus theo hadng din cua nhfi san xu^Jt Kit. 5 ul RNA dupc taeh chi^t tii miu benh ph&n cua Ipn nghi m^c PRRS dupc txdn vdi 20 ul hon hpp RT-PCR eua bp kit One step RT-PCR kit (invitrogen). Cfip mdi dUdc sb dung cho phan ling RT-PCR gdm mdi xudi 5'- CAGCCAGTCAATCAGCTGTG - 3' vfi moi ngupc 5'- TCGCCCTAATTGAATAGGTG - 3' nhfim khuyech dai doan gien 0RF7 dfii 392 bp.
Tien hfinh phan iing khuech dai san phfim trong may PCR vdi 35 chu k^. D i ^ di Idem tra ket qua RT-PCR b hi$u dign the lOOV trong 30 phiit. Quan sat ket qua dien di san p h ^ RT- PCR tren may chp.p anh gel vfi chup anh.
3. KET QUA v A THAO LUAN 3.1. Tri^u chufng lam s a n g c h u y^u ciaa 1 ^ con mftc P R R S
Bang 1. Trifu chiKng lam s a n g chu yeu ctia 1 ^ con m a c P R R S STT
1 2 3 4 5
Kl hi^u Ll L2 L3 L4 L5
Sit +++
++
+++
+++
++
Chfin fin, bdfin
+++
++
++
+++
++
Phfit ban +
Tn$u chung Ifim sfing Mf mat Kh6
sifng thif ++ ++
+ ++
+ + +
Tim tai +++
++
+ ++
Chfiy nu'dcmQi
+ + +
Tifiu chfiy +++
++
+++
+++
++
Tfio bin ++
+ + ++ + +++ N$ng; ++ Trung binh; + Nhe; - Khong bi^u hi^n
Nguy§n Thi Lan. NguySn Thi Hoa
B a n g 2 . Bife'n d o i d a i t h e c u a Itfn c o n c a i sufa m a c P R R S
Phoi Sung h u y i t X u l t huygt D|ch vi§m trong I6ng p h i
qufin
Sung h u y i t M^t c i t l6i c h i y djch
mSu h6ng
X u l t h u y i t
<3ilm Lo6t il mfim m^c ru^t gig
Ldch nh6i huyit, Gan ho'i si^ng
Viem mau do xam
Cd diem hogi tu' Sirng to. th^m tim X u l t h u y i t trSn Xoang bao tim tich b^ m^it niSm nu'6'c
mac NSo xult huyit X u l t huy£t. viSm mfiu do
xdm, dich phCl trong I6ng p h i quan
X u l t h u y i t . s u r g to X u l t h u y i t
mdu dd mgng d i l m lan trfin m S r „ ' Z L4chnh4,huy4l mong, nrem ' mgc bong tr6c Co-timnhao p h l i cSng ph6ng, na tCi
n h i l u 6 vi6m rai rSc trfin b l Thuy thOng, su'ng to m j t , x u l t h u y i t pliAi
X u l t h u y i t ailm X u l t huyit
Vifim mdu d6 xdm, mdt c i t I(h6 ho'i l6i
Khflng c6 bfinh tich
Nifim mgc thuy
thQng, c6 v i t Khfing c6 bfinh tich
Hinh 1. Phdi vigm, xu^t huy^t Hinh 2. Thfin xuAt huyet di£m
B a n g 3 . B e n h t i c h v i t h g ' c u a l d n c o n c a i s i ^ a m a c P R R S
Lpn
Ll L2 L3 L4 L5
Xuat huyit +
Phil Vifim l<e phli
+
Phi quan phi vifim
• +
+ HLB Xult huyit +
+ 00* quan
Ldch Nhli huyit
+ Hogi
tip +
+ Th|n Vifim clu th$n
Ru^t Thdm nhiim te
bdo vifim +
+
[J: HLB: Hach l a m b a + : Co b e n h tich - : Khong co b e n h tfch
Chan doan hOi cliu'ng roi loan h6 hap v^ smh san (PRRS) a Ign con .. va ky thuat PCR
Qiinn .sat l(in con cat sdii tronj,' do Lum Ur li - 6 tiiiin nghi niAc I'RKS d mtU ao gia t r a i tni H a n g Yen, Hai D u d n g , B.^c N i n h clio t h a y lpn con cai sfln nuic P R R S c6 cac t r i p u c h d n g l a m sfing c h u y^'u ifi sd't, c h a n fin, hd fin, t i m t a i , k h d thd. t i e u c h a y , t a o bdn t r o n g dd hipn t u p n g sd't, c h a n fin, bo fin cd m d c dp nfing d tfi't ca cac ldn t h e o ddi, d a y lfi cac dfi'u h i e u b e n h ly t h a d n g gfip t r o n g eiic bpnh t r u y e n n h i o m va k h d n g cd t i n h dfic t r a n g ( B a n g 1). H i c n tUpng tim t a i . thci Ithd. tiuu c h a y , t a o bdn c6 bigu h i e n d m d c dp t a trunj^
b i n h ddn nfing, d a y cd t h e dUdc coi lfi nhQnfj dfl'u hieu diiu t i e n cho p h ^ p c h u n g t a n g h i d e n lpn m d c P R R S . Cac b i e u h i c n k h a c n h u c h a y nUdc n m i , mi mfit s u n g , p h a t h a n , xu;l't h i e n rai r a c d cac ldn k h a c n h a u va c6 m d c dp n h e d e n t r u n g b i n h .
3.2. B i e n d o i b e n h Iy c u a Itfn c o n c;iii s i J a m f i c P R R S
3.2.}. Bien ddi dni ihc cua hm con cai .siia miic PRRS
Benh tich d a i t h e c u a ldn miic P R R S x u a t hien cf n h i e u cd q u a n k h a c n h a u ( B a n g 2). T r o n g dd, ta't ca cac t r U d n g h d p m o k h a m d e u CO b e n h tich d phoi. P h d i viem vdi cac miic dp k h a c n h a u t u y t h u p c v a o tufng ldn b e n h va miic do t i e n t r i e n c u a b e n h c d n g
n h u .sa lie phiU cac b e n h k h a r , . .'in khong m a n g t m l i cliat dd'i x i i n g . C a c t l i u y phoi bi viem t h a d n g cd m a n dd x a m , co t r U d n g hpp phdi bi n h u c h d a . n h a t m a u , xufi't huye't tren bd mfit p h o i . Phdi v i e m vfi p h i i l a m cho cac t h d y phoi ciing lai, m $ t cfit hdi loi ( H i n h 1).
C a c dfi'u hipu nfiy c6 t h e d a p c giai t h i c h rfing Ipn s a u cai sUa n h u cfiu t r a o doi chfi't cao din d e n n h u cflu oxi tfing len, t r o n g k h i dd virus P R R S lai x a m n h d p va n h a n len t r e n cac dai t h u c bao phoi dfin de'n phoi v i e m vfi lam cho lpn c a n g khc3 t h d h d n . K e t q u a nfiy la phii h d p vdi bno cao c u a P h a m Ngpc T h a c h , Dam Vfin P h a i (2007).
T r o n g quii t r i n b m o k h a m q u a n sat b e n h tich d a i t h e , lpn con mfic P R R S thupng thA'y cac h a c h l a m ba t u m a u sUng to. tu m a u n h i e u nhfi't la c a c h a c h l a m ba viing jDhoi. Q u a n s a t cac h a c h n a y d e u tha'y hach Lu hdn b i n h t h U d n g .'3 - b ! a n . m a u do thfim, CO t r U d n g h d p xua't h u y e t , cat doi hach co dich m a u h o n g c h a y r a .
Thfin xufi't h u y e t d i e m la b e n h tich hay gap cd n h i e u d i e m xufi't h u y e t t r e n be mat t h a n va ca t r o n g m o thfin. Q u a n s a t con thay h i e n tUpng t u h u y e t d t h a n , n h u n g bie'n doi n a y chi thfi'y xua't h i e n t r o n g mdt s o i t trUPng h d p ( H i n h 2)
f
Hinh i. Djch phii Irong I6ng phe nang Hinh 4. Lfich xuat huyel
Nguy§n Thj Lan. Nguyin Thi Hoa
Hinh 5. Ket qufi phfin irng RT- PCR
[Vu-us PRRS dupc phfit hi#n bling phan img RT- PCR vdi do dai cua gen la 392bp, thang chuin M lOObp; gig'ng tii 1-10 lfi mSu tilt 5 lpn blnh theo thii tU bang 4; gieng 11 la doi chiing am; gig^ng 12 la doi chiing dudng]
Cfic benh tich dai the khfic thay doi tu^
thupc vfio dien bie'n eua binh ciing nhU sU ke phat cfic benh khac.
3.2.2. B?nh tich vi thi cua Ign con cai sua mdc PRRS
Benh tich vi the dang chii J nhfi't ciia ca 5 ldn dupc nghien ci3u b tren la nhiJng biS'n do'i b phd'i. Phe nang chiia diy dich ri viem va dai thuc bfio (DTB), quan sat thfi'y xufi't hiin te bfio khd'ng Id nhilu nhan. Cac tieu ban vi the con thfi'y mdt dfic trUng nfla la sU thfim nhiem cua te bfio phe nang loai II
(pneumocyte) Iam cho vfich phd' nang nhfin Iai, thudng bat gap dai thuc bao phan buy trong phe nang. Phoi xuat huyet ro long ph^
quan va phe nang chUa day hdng ciu mau do tUdi. Nhilu t^ bao phdi bi hoai txi vfi cd huyit khdi nho trong long mach quan do cac thfinh phan mau tach ra va dong lai phe nang tran ngfip dich phu (Hinh 3).
Lach sung huyit, thoai hoa va hoai td.
Tren tieu ban lach vach ddt nat, cac te bao thoai hoa xen ke vdi cac t l bao lfinh, thfim nhiem hong cau lan trfin trong nhu mo lach (Hinh 4).
Bang 4. Ket qua phan ifng RT- PCR xac dinh virus PRRS Lqm
Ll L2 L3 L4
L5 Dli chi>ng fim Dli chirng dirang
Lopi b^nh phim Phir Ri>mdt H^ch phli
Phoi Phil Nird'c mui
Th^n Nird'cmGi
Phli Ru$t Mlu nirdc cit Mau RNA cua PRRSV
Kit qufi RT- PCR
*
*
+ +
+
+
Chin doan hpi chLpng r6i logn h6 h^p va sinh s^n (PRRS) 6- ipn con . va kg thugt r PCR Thfin ngofii nhiing biln doi b^nh 1^ nhU
thfim nhilm te bfio vifim, t l bfio th^n bj thoai hoa, ho^i tii thi cdn cd nhflng biln d^i b kS thfin nhu xufi't huyl't, t(lp trung nhilu hdng cfiu vfi cfic lymphocyte, monocyte...nlu qufi trinh vidm kdo dfii thi tfing sinh nhilu fibroblast. Cfiu th§n viem cd chiia dieh ri vigm, t l bfio 6'ng th^n teo nhd, Idng dng rdng, c6 try trong. 6 cfic viing lfinh c6 ho^t dOng bd, mach quan gian rdng, phfin nhilu nhfinh.
3.3. Ket qua p h a n iing RT- PCR Ta cac mfiu b^nh phfi'm, RNA ciia virus dupc tach chilt bfing kit tfich chilt QIAamp. San phfi'm thu du^c sau khi tfich ehilt dUde khuleh dai bfing ky thu^t RT- PCR vdi sa tham gia cila enzym sao chdp ngupc (Reverse Transcrip) vfi sii dung cfip mdi 0RF7 cd kha nfing phat hien virus PRRS thupc ca chung bfic My vfi ehung chau Au (cfip mdi nfiy cho p h i p xac dinh doan gen ciia virus PRRS ed kich thUde 392bp). San ph^m sau khi khuech dai dUdc dien di va chup anh. Ket qua ciia phan ling RT- PCR xac dinh PRRSV dupc trinh bfiy b bang 4 va hinh 5.
Kit qua di^n di kiem tra san ph^m cho thfi'y tfi't ea 10 mfiu dupc chpn lfim thi nghiim diu cho kit qua dUdng tinh vdi PRRSV. San phftm dien di eho vach DNA tUdng ling 392bp dung theo thiet k l moi. Cac mfiu benh ph^m bao gom: phd'i, h^eh pho'i, nudc miii, rii mfit diu cd the sii di^ng Ifim mfiu de ch^n doan PRRSV.
4. KET LUAN
Chfl'n dofin hhng ky thufit binh ly cho thfi'y cfic tri^u chiing lam sfing chu yeu cua I^n sau eai siia mfic PRRS bao gdm: sot, bo fin, khd thd, tfm tai. B§nh tfch dai t h i tfip trung chii ylu b phoi vfi hach lam ba vdi hi^n tupng vidm lfi phd' biln. Cac biln dli vi t h i ph6 hii'n b phoi vdi hi^n tUpng tham nhilm t l bfio vidm, cfic cd quan khae nhU h$ch, lach, th$n cung xufi't hi$n cac t^n thUdng vi t h i . Bfing ky thufit RT-PCR, da xac dinh dupe chfnh xac dU^c l0n sau cai siia m^c PRRS.
TAI LIEU THAM KHAO Ph?m Ngpc Th^ch, Dfim Vfin Phfii (2007). "M^t
sd chl tifiu Ifim sfing, chl tiSu mfiu d l?n mfic hpi chiing rd lo^n sinh sfin vfi hd hSp (b^nh tai xanh) trfin mpt s6 dfin Ipn t^ii tinh Hfii Duong vfi Himg Yfin", Hpi thfio Hpi chung r6i lo^n hd hdp - sinh sfin vfi b^nh lien cau khudn d Ipn, ngfiy 11/10/2007, DH Nong Nghidp Hfi Ndi
Cavanaugh, D. (1997). Nidovirales: a new order comprising Coronaviridae and Anerivtridae.
Arch. Virol. 142:629-633).
Chung, W, B., W. F. Chang, M. Hsu, and P. C.
Yang. (1997). Persistence of porciiK reproductive and respiratory syndrome virus in intensive farrow-to-finish pig herds. Can. J.
Vet. Res. 61:292-298.
Done, S. H., D. J. Raton, and M. E. C. White.
(1996). Porcine reproductive and respiratory syndrome (PRRS): a review with emphasis on pathological, vi-rological and diagnostic aspects. Br. Vet. J. 152:153-174.
Keffaber, K. (1989). Reproductive feilure of unknown aetiology. Am. Assoc. Swine PracL Newsl. 1:1-10.
