KHOA HOC KY THUAT THU Y TAP XX S 6 3 - 2013
UTNG DUNG KY THUAT HOA MO MIEN DfCH CHAN flOAN BENH DjCH T A LQfN
Biti Iran Anh Ddo, Nguyin Thf Lan Khoa thu y, Trudng Dai hgc N6ng nghi?p Hd Ngi T O M T A T
Nghien ciiu nhdm irng dung ky thuat hoa mo mien dich vdo chdn dodn b^nh djch td Igm. Tir 10 Ipn CO nhiing biau hien ve mat lam sang cua b?nh djch ta Ign, chiing toi ti§n hdnh mo kham vd lay cac mau b?nh pham phyc vu cho nghian ciiu. Ket qua nhupm hoa m6 mien dich cho thay cd 10 Ign deu duong tinh vai virut dich td Ign, trong do hach amidan, lach vd hach mdng treo rupt c6 ty le duong tinh Id 100%; mgt non vd gan c6 ty le duong tinh la 80%, con lai cdc mo khdc co ty 1? ducmg tinh thap hem vd thdp nhat Id 6 ruot gia, chi dat 30%o. K6t qua nghien ciiu cho thay rdng cac mau mo nhu hach amidan, Idch, hach mang treo rugt la nhirng co quan t6 chirc co dg chinh xac cao khi tiln hdnh chdn dodn benh djch ta Ign bang phuong phap nhugm hoa m6 mien djch, tir do giiip cho vi?c lay mau phdn lap virut hi thuc dia.
Tie khoa: Dich td Ign, Chan dodn, Hoa m6 mian dich
Application of immunohistochemistry technique for the diagnosis of Classical Swine Fever
Bui Trati Anh Dao, Nguyen Thi Lan
SUMMARY
The research apphed immunohistochemistry technique to diagnosis of classical swine fever (CSF) in pigs Ten pigs with clinical sign of CSF were necropsied for taking samples to sen/e for this study. The results showed that ten pigs were positive for classical swine fever virus, in which the positive rate of tonsils, spleen and lymph nodes in mesentery were 100%; in small intestine and liver were 80%, another tissues had lower positive rate and lowest positive rate in the colon were only 3 0 % . The results of this research indicated that tonsils, spleen, lymph nodes m mesentery were exact for diagnosing by immunohistochemistry and useful for isolating virus from field.
/Ceytvords; Classical swine fever, Diagnosis. Immunohistochemistry
I. D A T V A N D E khong xay ra cac 6 djch Ion vd gdy thiet hai nhir n,^u t-i I™, /D ,• 1 1- -* L- L truac nhung vdn luon la moi de doa cho nganh Dich ta Ign (Pestis suum) la mgt benh .„ ; . . / , . 7 , , . truyen nhiam nguy hiam do virut thugc nhom
pestisvirus - hg Flaviridae gay ra tran Ign (Jubb Kennedy vd Palmer, 2006). Benh xudt hien 6 mgi lira tuoi ciia Ign voi t6c do lay lan nhanh vd manh, 6 the cdp tinh benh gay chet nhiau Ign vcfi ty le tii vong cao hr 90 - 100%. 6 Viet Nam, benh dich td Ign da gay thiet hai nghiam trgng d6i VOI nganh chan nuoi Ign vao cdc thap ky 70, 80 va 90 ciia tha ky 20. Hien nay benh Uiy 12
chan nu6i, do do viec chdn dodn nhanh va chinh xdc benh dich td Ign la rdt cdn thiet. Myc dich ciia nghien ciiu nay nhdm thii nghiem phuong phdp hoa m6 miln dich (Immunohistochemistry - IHC), mpt trong cac phuong phap chdn doan voi nhirng uu diem nhu co dp nhay, dp chinh xdc cao va dac bi?t cho phep xac djnh sv phan b6 cua virut a mo bao, trong h6 trg chdn doan chinh xac banh dich td Ion.
KHOA HOC K? THUAT T H C Y TAP XX S 6 3 - 2013
II. VAT LIEU VA PHlTONG PHAP NGHIEN ClTU
2.1. V§t lieu
10 Ign nghi mdc b$nh dich td thupc cdc gia trai viing phu can Ha Ngi {cdc Ion nay chua dugc tiam vacxin phong benh dich ta).
Cac Ign nghi mdc DTL dugc m6 khdm da Idy mdu. Cdc mau m6 cua Ign bSnh gom: ndo, ph6i, tim, gan, lach, than, hach amidan, hach mdng treo rugt, ruOt non, rupt gid.
2.2. Phirffng phdp nghien cii-u
- Su dung phuong phap quan sdt, th6ng ke sinh hpc: True tiep quan sat cdc trieu chiing lam sdng cua lan benh kat hgp diau tra thu thdp cdc th6ng tin tir chii gia trai va can bp thii y dia phuong de thu thap mdu Ign benh
- Ky thuat RT-PCR chan doan cac Ign nghian cim mdc dich td Ign.
- Su dung phuong phdp hoa mo mien dich d6i VOI cdc mdu mo: Diing phdn ling khdng the - khdng nguyen (KT - KN) ddc hieu d6i voi cdc manh cat da chuyen due paraffin. Cdc mdnh cat sau khi da khu sach paraffin dugc phii KT lan tran be mat. Nau trong m6 benh pham co KN tuong img vdi KT, phirc hgp KN-KT se dugc hinh thdnh. Phiic hgp nay dugc nhdn biet nho he th6ng khuech dai tin hieu bao gom KT bdc cau (khdng KT) vd hod chdt nhugm mdu DAB (2,3 - Diamtnobenzidin).
Cac buac tien hdnh ky thuat hoa mo mien dich dugc thuc hi8n theo qui trinh tai Phong thi nghiem trung tam, Khoa thii y, Truong Dai hgc Nong nghiep Hd Npi:
+ Bu-dc 1: lam sach tiau ban
Khii paraffin, khu xylen, khir c6n gi6ng phuong phdp lam tieu bdn vi the. Sau khi cho chdy duoi voi nuac, nia lai tieu ban bdng nucrc cat.
+ Birdc 2: boat hod enzym
Ngdm ngap tieu ban trong dung dich PBS va hdpu6tal21''C/5phiit.
+ Birdc 3: khir peroxydase npi sinh Diing H,Oj trong dung m6i Methanol (1 H^Oj 30%: 9 Methanol). Ngdm tieu bdn Irong 10 phut.
+ Budc 4: gdn khang the
Nh6 80 )il KT DTL \hn tieu ban. Da hi dm 37''C/lh hodc 4''C/qua dam. Rua tieu bdn bdng dung djch PBS 3 Idn (5 phiit/ldn).
+ Budc 5: gdn khdng khang the Nh6 SO^il khdng khdng the len deu bdn. De hi dm ST-C/lh. Rua PBS 3 lan (5phut/ldn).
+ Budc 6: cho ca chdt
Ngam tiau ban trong dung dich DAB khoang 5-8 phiit.
Kiem tra duoi kinh hien vi thucmg. Neu thdy bdt mdu, dimg phdn ling bang nuoc sach. Neu kh6ng, ngam tiap tiau ban trong DAB den khi xuat hien mau.
+ Buac 7: nhupm nhan ta bao Nhupm bdng hematoxilin (30s), Idm sach, gdn lamen vd quan sat bang kinh hien vi quang hgc.
Khi CO mat ciia virut trong t6 chiic dem lam hoa mien dich se cho ket qua duang tinh vai su hien mdu ndu vdng tren lat cat to chiic (mau ciia DAB). Virut tap trung cdng nhieu, bat mdu cdng ro, mire duang tinh cdng cao. Yen moi 16 hod m6 mian dich, dau co d6i chiing dm tinh la Idt cdt m6 ciia Ign khoe.
III. K E T Q U A K H A O N G H I E M 3.1. Ket qua thu thdp mau nghien ciru
Cdc mau Ign nghi mac dich ta a viing phu can Hd Ngi dugc thu thap dua theo thong tin ciia cdn bg thu y, chii gia trai va dugc kiam tra mpt so trieu chirng lam sang chii yau ciia Ign nghi mac dich td. So mau thu thap vd cdc trieu chimg lam sang chii yau dugc trinh bdy tai bdng 1.
KHOA HOC KY THUAl THLJ Y lA? XX S6 3 - 2013
Bdng 1. M l u i^n nghi mac djch tk thu t h | p diT^c
Nhom 1 2 3
D5i tU0ng Lpn con theo m§
LOn sau cai sQa L0n choai Tong s6
S6 lUOng (con)
9 11 5 25
Tri$u chijTng Idm sang chu y^u S6t cao, c6 dii m^t, run r i y , nlim don d6ng, co gidt, fa phdn tr^ng, phdn loang mdu vang thdi kham, xudt huySt di^m vimg da m6ng
Sdt, b6 dn, I0n ndm d6n d5ng, xuSt huydt dilm vung da mfing, phdn tdo sau loang vang thfii kham Sdt, b6 dn, nSm d6n ddng, mi m^t sung, chdy nude m^t, tdo bdn, Idng XLJ
Lgn nghi mac benh dich ta co cac trieu chiing lam sang chii yeu la: sot cao, b6 an, r^t run nam ddn d6ng, lgn han dau tao b6n roi ia chay manh, xuat huyat diam a cac viing da mong nhu b6n chdn, bung, tai. Mgt s6 co biau hien tho kho vd CO trieu chimg co gidt. Trong cac trieu chiing tran, s6t cao, bo an, ret run nam don dong, tao bdn Id cdc dau hieu benh ly thuimg gap. Cac trieu chiing lam sang cua lgn mac benh dich ta trong nghien cim nay phii hgp voi cac mo ta ciia mgt so tac gia trong va ngoai nuoc (Bin Trdn Anh Dao va Nguyan Hiru Nam, 2009; Ddo Trgng Dat va Nguyen Tian Diing, 1990; Jubb Kennedy vd Palmer, 2006; Nguyen Tdi Nang, 2007). Ngoai
ra, s6t con lam cho vat mat nuoc, dnh huong den nhu dgng rugt khian lgn hi tdo bon. Cac tri^u chirng nhu: xudt huyat diam a viing da mong, Ign ia chay manh, phan th6i khdm xudt hi&i voi tdn suat it hoac nhieu co the la cdc dau hieu dau tian ciia benh dich ta lgn (Biii Tran Anh Dao va Nguyln HiJu Nam, 2009).
3.2. Ket qua nhuom hoa mo mien djch Tir cac mdu lgn benh thu thdp dugc, chpn ngau nhien 10 lgn de mo kham, Idy mdu cac ccr quan gom: nao, gan, than, lach, tim, phoi, hach lympho, rugt non vd rugt gid. Kat qua nhupm hod mian dich dugc trinh bdy o bdng 2.
Bdng 2. Ket qud u'ng dung ky thuat tioa mo mien djch vao chan doan b^nh djch td Icfn Mo nghien ciJu
Hach amidan Lach
Hach mang treo mot Rupt non Gan Than Phoi Nao Tim Ruot gia
Sd' m l u nghien ciJTu 10 10 10 10 10 10 10 10 10 10
Sd' m l u dUdng tinh 10 10 10 8
a
6 5 4 4 3
Ty i$ dUdng tinh (%) 100 100 100 80 80 60 50 40 40 30 Kat qua 6 Bang 2 cho thay, 10 mau dau cho
ket qua duong tinh vai dich td lgn. Nhung kat qua nhugm hoa m6 miln djch 6 m6i mo la khac nhau. O hach amidan, lach vd hach mang treo
rugt, ty le duong tinh la 100%. 6 ru6t non va gan ty le duong tinh Id 80%, con lai cac mo khdc CO ty le duang tinh thdp han, ty le ducmg tinh thdp nhdt Id 6 ru6t gia 30%. Theo chiing 14
KHOA HQC Kf THUAT THU Y TAP XX SO 3 - 2013
t6i, nguyen nhdn ciia hiSn tugng nay la do hach lam ba va lach la dich tin c6ng ciia virut. Virut tang sinh manh a hach vd Idch roi lan sang cac CO quan khdc. Do do ty le duang tinh cua hach mang treo rugt, hach amidan va Idch khi nhupm hoa m6 mian dich la 100%).
Cdc mo khac ciing cho ty le duang tinh cao Id nipt non, gan, than vd phoi voi ty le duong
tinh Id tir 50 - 80%) s6 mau nghian ciiu. Theo chiing toi cac ca quan nay c6 t6c dp luu th6ng mau Ion, h? th6ng mach qudn phdt triln va cd so lugng Idn cdc te bao thuc bao nhu: cac te bao lympho 6 mang Payer, cdc dai thuc bdo pha nang d ph6i vd cdc tl bao Kuffer 6 gan... Cdc tl bdo ndy cd khd ndng thirc bao rat Idn. Virut nhan len rat nhanh trong nguyen sinh chat ciia nhiing te bao nay.
Hmh 1 Virut phan bo d lach (IHC X 60)
Hinh 2 Virut trong te bao niem mac ruot (IHC x 600)
i if
V "^Hinh 3. Virut phan bo d hach mang treo rugt (IHC x 150)
Hinh 4. Virut phan bod than (IHC X 150) Mat khdc khi quan sat cdc tieu bdn tren
kinh hian vi quang hoc cho thay su phdn bo cua virut dich ta lgn trong cac to chiic nhu sau:
6 hach amidan, virut phan b6 rai rac khap vi trudng trong cdc dai thirc bdo, te bdo bieu md hoc amidan vd te bao ngi mang amidan. 0 lach, virut phan bd rdi rdc khdp cac vimg tuy do vd tuy trang cua lach. Dac biat virut tap trung cao
thanh dam Idn bdt mdu ndu ddm ciia DAB 6 cac vimg nhdi huyet va trong cac ddng mach biit Idng. O vat kinh x 40, virut dugc tim thdy trong ta bao dai thuc bao va te bao ludi ngi bi. 0 hach lympho mdng treo rupt, virut phan bd rdi rdc trong cdc nang lympho, trong cdc dai thuc bdo vd te bao lympho. 0 rugt non, virut dugc tim thdy trong te bao niem mac ciia rugt vd rdi
KHOA HOC K"? THUAT THC Y TAP XX SO 3 - 2013
rac trong cdc dai thuc bdo, te bdo lympho ciia mang Payer. CJ gan, virut dugc tim thay rai rdc trong cdc tiau thuy gan, trong cdc ta bao Kuffer va ta bao npi mac ciia mao quan. 6 ph6i, virut d trong cdc ta bao bieu md ciia nhanh phdi vd cdc dai thuc bdo phe nang. O than, virut dugc phdt hien trong cdc te bao dem nhan, ta bao bieu md 6ng lugn va ong gdp. O tim, virut cd trong ta bdo ngi mac ciia mach quan va sd lugng it trong te bdo CO tim. Ci ndo, virut dugc tim thdy rdt it trong te bdo ndi mac ciia mach quan d viing cd su tham nhidm cdc te bdo viem. O rupt gid, ty 1?
duong tinh thap nhat (30%o), virut dugc tim thdy trong cdc ta bao niam mac ciia manh trang.
Nhu vay kat qud nhugm hod m6 mian dich dd cho thdy virut phdn bo va tap trung chii yau d cdc t6 chirc nhu: hach amidan, hach lympho vd Idch . 0 rugt non, gan, than, virut ciing xudt hien vdi ty le thap han, cac to chirc khac virut xudt
hien vdi ty 1? thap hon. Qua day cho thay, khi irng dyng ky thuat hod mo miln djch vdo chan doan h$nh dich td lgn, nen Idy mo h^nh phdm la hach amidan, hach mdng treo rugt vd Idch. Cac mau mo ciia gan, phdi, rugt non va than khi lam hod m6 mien djch cung co y nghTa trong chdn doan h^nh.
Ngodi ra mat dp tap trung ciia vimt d cac mo bao cd the ddnh gid theo mat dp bdt mdu duang tinh d cac tieu ban vi no tuong img vdi mat do tap trung ciia virut d Idt cdt md ciia tiau ban dd.
Mat dg bdt mdu ducmg tinh nhieu, xudt hien khdp vi trudng; mat dg virut t^p trung cao (+++). bdt mdu vira phdi la mat do virut tap trung miic trung binh (++). bdt mdu it Id mat dg virut tap trung ft (+). Khdng cd bdt mdu duong tinh: phan ling dm tinh, khdng cd mat ciia virut (-). Ket qud ddnh gia mat do tap trung ciia virut dich ta lgn trong cdc to chirc dugc trinh bdy d bdng 3.
B^ng 3. Sii phan bo va m^t dp cua vhut DTL trong cac cd quan cOa Idn m^c b^nh dich td
Mdu \0r\~~~~-,..__^^
LI L2 L3 L4 L5 L6 L7 L8 L9 L10
H^ch amidan
+++
+++
++
+++
++
+++
+ ++
+++
+++
H^ch lympho
+++
++
+ ++
++
++
+ + ++
+++
Lach +++
+++
+ ++
++
++
+ + ++
++
Gan ++
++
+ + + +
++
++
Rupt non ++
++
+ + + +
+ ++
Th$n ++
+
++
+
+ ++
Ph^i ++
+
+
+ +
Nao + +
+
+ Tim
+ +
+
+ Ru^t
gia + +
+
Ghi chu- (+++): Mat do virut tap trung cao; (++)• Mat do virut tdp trung vira phdi;
(+): Mdt do virut tap trung it; (-): Phdn ung dm tinh khong cd mat cua virut Nhu vay cd the kat luan rang, cdc md bao cd
ty le duong tinh cao khi nhupm hoa md mien dich ciing tuong ung se cd mat do virut tap trung nhieu. O Ign mac benh dich ta, cac td chirc cd mat do virut tap trung nhieu la: hach amidan, hach lympho, lach, rugt non, gan va than.
Da danh gid dp nhay ciia ky thuat hoa m6
miln dich, chiing toi so sanh ket qud 1dm hoa md midn dich vdi ket qud chan doan bdng ky thuat RT - PCR.
Kat qud cho thay khi sii dung phuong phap hod md mian dich trong chan dodn b?nh 6 cac loai m6 khdc nhau cho ty le rat khac nhau, tir 30 - 100%.
16
KHOA HQC K? THUAT THtl Y TAP XX S6 3 - 2013
C) hach amidan, hach lympho va lach, 100%
m^u nghien cihi dlu cho kit qud duang tinh vdi virut DTL tning vdi kit qua lam RT-PCR. Trong khi do 6 nao, tim va rupt gid chi phat hi?n 30- 40%) mlu cd vhut. Dilu nay cd the dugc giai thich la do su phdn bd khong d6ng deu cua virut trong ca the lgn benh. () cac t6 chiic thich hgp cho virut nhan lan vd phdt triln hoac la dich tan cdng cua virut thi virut sS tdp trung nhilu 6 do vdi tan suat Idn, ngugc lai vdi cdc t6 chirc it
tuong thich, virut sg xudt hi^n it vdi tdn sudt nhd. Kat qua ndy cho thay dg nh^y ciia ky thuat hoa md mien dich phy thugc rdt Idn vdo viec chpn td chirc md dem chdn dodn.
Nghien ciru cua chiing tdi cung cho thay, khi lam hod m6 mien dich, ngoai uu diem quan sat dugc sy phdn bd cua virut trong cdc m6 ciia lgn b?nh, kit qua 1dm hod md miln djch d hach amidan, hach lympho vd Idch ciing chinh xdc tuong duong vdi ket qua lam RT-PCR (Hinh 5).
Hinh 5. K0t qui tJi$n di san phim phan Ctng RT'PCR
(Thang chudn Marker: WObp; gi€ng tit 1-5 la 5 miu benh phim duoc lay tddng Ling d 10 Idn nghien cdu; gii'ng 6 la dSi chOng Sm (DW);
gid'ng 7 M doi chiing duang).
IV. K E T L U A N
- Su dung phucmg phap nhugm hda md mien dich xac dinh dugc 10 lgn duong tinh vdi benh dich td lgn, trong khi dd ty 1$ duang tinh xdc dinh dugc d hach amidan, Idch vd hach mdng treo rupt Id 100%, d rupt non vd gan Id 80%), cac mo khac cd ty le duang tinh thap hon, thdp nhat Id d rupt gid (30%)).
- De cd dp chinh xdc cao khi tien hdnh chan dodn b?nh dich td lgn bdng phuong phdp nhugm hda md miln dich hodc Idy mlu phan lap virut dich td Ion, cdn lay cdc mdu m6 hach amidan, Idch hodc mang treo rupt.
- Phuong phdp hda md miln dich cd dp chinh xdc cao, de lam va cd gid thanh hgp ly, cd thi dp dung tai Phong thi nghiem trung tam Khoa thii y d6 chan doan benh dich ta Ign.
TAI L I E U T H A M K H A O 1 Bill Tran Anh Dao, Nguyan Hiiu Nam. 2009.
Mdt sd dac diem benh ly d lgn mac benh dich td. Tap chi Khoa hoc va phdt trien 7(2): 166- 171.
2. Ddo Trgng Dat, Nguyen Tien Dung. 1990.
Tinh hinh dich tl cua benh dich td lgn d Viet Nam vd vdn de phdng chdng. Ket qua nghian ciru khoa hpc vd ky thuat thii y 1979- 1984:5-10.
3. Jubb Kennedy, Palmer. 2006. Pathology of Domestic Animals.
4. Nguyln Tai Nang. 2007. Dien bien lam sang benh dich td Ign tai Trai Ign nai ngoai xa Son Duong (Lam Thao - Phii Thp). Tgp chi Nong nghiep vd Phdt tnen nong thdn: 91-92.
Nhan ngay: 21.1.2013 Phan bien ngay: 7.2.2013
