KHOA Hpc K? THUAT THO Y TAP XX Stj 2 - 2013
tfNG DUNG KY THUAT PCR TRONG CHAN DOAN RENH HO HAP MAN TJNH (CRD) DO Mycoplasma galliseplicam TREN GA BAN DjA
Nguyen Bd Hiin, Le Vdn Phan vd Tg Kim Chung Khoa thil y - Dgi hgc Nong nghiip Hu Ngi
T6IVI TAT
Ky thuat Polymerase Chain Reaction (PCR) sir dyng cdp moi ddc higu MG14F vd MG13R cho Mycoplasma gallisepticum (MG) theo cdng bd ciia OIE (OIE., 2008) da dugc ung dung thdnh cdng trong chdn dodn benh CRD do MG gdy ra Iran gd ban dja. Ket qua chdn dodn bdng ky thudt PCR cho thdy 20/43 (46.5%) mau bgnh pham Id phdi va khi quan ciia gd nghi mdc bgnh CRD cho kat qua duang tinh vdi MG. Sdn pham PCR ddc higu thu dugc cii kich thudc khodng 185 bp. Trong nghien Cliu ndy. mdu phdi ciia gd nghi mdc bgnh CRD cung dd dugc sir dyng da phdn lap MG. Ket qua phan lap MG tren mdi trucmg phdn lap Mycoplasma Agar (MA) vd Mycoplasma Broth (MB) cho thay 89/174 (51,15%) mau bgnh phdm cho ket qud duong tinh tren mdi trudng MB, trong khi 72/174 (41,38%)) mau bgnh pham cho ket qud duang linh Iran moi trudng MA.
Tirkhda: Gd, Benh hd hap man tinh (CRD), Mycoplasma, PCR
Application of technique for detecting chronic respiratory disease (CRD) caused by Mycoplasma gallisepticum in domestic chickens
Nguyen Ba Hien, Le Van Phan and Ta Kim Chung
SUI\/II\/IARY
Polymerase Chain Reaction (PCR) using MG14F and M G I 3R specific primers for Mycoplasma gallisepticum (MG) according to the promulgation of OIE (OIE., 2008) has successfully been applied for detecting chronic respiratory disease (CRD) caused by MG in domestic chickens.
The diagnostic results for C R D by using PCR technique indicated that 20/43 (46.5%) of trachea and lung tissue samples of chickens suspecting Mycoplasma gallisepticum infechon were positive. T h e size of specific PCR product was 185 bp. in this study, the lung tissues samples of C R D suspected chickens were also used for isolation of MG in Mycoplasma Agar (MA) and Mycoplasma Broth (MB) medium. The studied results showed that 89/174 (51.15%) samples were positive for MB media while 72/174 (41.38%) samples were positive for MA media.
Key worcfe; Chicken, C R D , Mycoplasma, PCR
I . D A T V A N D E nudi gia cdm do Iam gidm san lugng trimg ddi
^ . . ; , , „. ,. , ^ , vdi gd da va san lugng thit ddi vdi gd thit. Cdc Vl khuan Mycoplasma gall-sep„cum (MG) 43, ailm trieu ching lam' sang chinh cua benh thupc ho Mycoplasmataceae^ ,B?"J.^RD do ^ ^ j _ . . _ ^ ,, ^^^^ ^.._^
MG gay ra la mot bgnh Viem duang ho hap man , , . °; ^, i. . . ^,. , . , ,,
^ •'^ , 1 . , ^ , . f . , , , . va nhfing ton thuang rat nang a tui khi (Ley, tinh a cac loai gia cam. MG chu yeu gay benh a -,Ani\ D - i. .-nr*» • - - *_• - - .
^ , , . . . ,: zJ, : ,^ 2003). Benh CRD tren ga se tra nen nang va gavaga tay, taynhienngucntacungda phan lap ^ ^ ^ ^^ ^. ^^^ ^ . ;J^^^
duac mam benh nay a ga loi, ga go, cong tn, ^.^ ^^.^ ^^^ Niucatxon, benh viem chim s6, chim cut, ga tSy hoang da, vit da dieu. ^^ x^ _^. j ^ (infectious bronchitis), Benh CRD gay thiet hai \(m cho nginh ehSn ^ ^ ^^ ^. ^_^.^ ^ ^ ^ , .
KHOA HQC K? THUAT THU Y TAP XX S 6 2 - 2013
MG cd th^ dugc chdn dodn bdng nhiau phuang phdp mien djch hgc khdc nhau nhu phan ung mien djch huynh quang. phdn dng ngung kel nhanh tren phien kinh, phan ung ELISA. phan irng ngdn trd ngung kel hong cau (HI), nhung nhO'ng plurang phdp ndy dugc cho Id CO nhieu ban che ve dO nhay vd d<> d^c higu (Klcvcn. 1998). Phan img PCR dd vd dang Id mgt phuang phdp dugc iya chgn vd dugc diing kha phd bicn trong cdc phdng Ihi nghigm dc chan dodn bgnh CRD do M(i Phdn irng PCR dugc cho Id phucmg phdp cd dg nhgy vd dg ddc higu cao han cdc phuang phap mien djch hgc, cho phap chan dodn nhanh vd chinh xdc cac chiing MG trong cdc mau bgnh pham nghi mdc bgnh CRD (Feberwee vd cs., 2005; Garcia vd cs . 2005; Ghoraibeh and Al Roussan, 2008;
Kempf vd cs.. 1993; Mardassi vd cs., 2005).
6 Vigt Nam, Nhij- Van Thy (2002) dd ung dung phuang phdp PCR trong chdn dodn nhanh vd budc ddu diau tra dich te bgnh do MG iren gd vd da dgt dugc nhihig ket qud budc dau ddng ghi nhan. De img dyng vd phat trian kJ thuat PCR thdnh mdt ky thuat thudng quy trong phdng thi nghiem da chan doan nhanh vd chinh xdc bgnh CRD do MG, chiing tdi ddt vdn de nghian cuu img dung ky thuat PCR trong chan dodn bgnh CRD tran gd bdn dia
II. VAT LIEU VA PHUONG PHAP 2.1. Thu thSp mSu b^nh pham
Benh pham: hgng, khi quan, phdi ciia gd nghi mdc bgnh CRD, mgt phdn dugc dem ra xet nghigm ngay de phan lap Mycoplasma vd vi khudn ke phdt cu tni d dudng hd hdp. Phdn cdn lai dugc bdo quan trong tu Ignh - 20"C dimg Idm nguyan ligu cho phdn ung PCR.
2.2. Chiing vi sinh v3t
Chung Mycoplasma gallisepticum (MG) va Mycoplasma synoviae (MS) chuan dugc diing trong phdn ung PCR Id chung ATCC 19610 vd ATCC 27399, hai chung ndy dugc cung cdp bdi Vien nghian c(tu thu y cua Hdn Qudc. Ngoai
ra trong nghian cuu ndy chiing tdi cdn sir dyng virul Niucatxon (LaSota) vd 3 chung vi khu^
gom Pmultocida. Salmonella vk E.coli Idm ddi chung am da kiem tra dg ddc higu cua phan irng PCR trong chdn dodn bgnh CRD do MG gay ra Iran gd. Mau bgnh phdm dugc diing trong nghian ciru ndy da chan doan MG la phdi vd khi qudn gd nghi bj bgnh CRD.
2.3. Phfin lap Mycoplasma gallisepticum Mdl trudng de nudi cay va phan lap MC dugc su dyng irong nghian ciru nay la moi trudng MB vd MA, ddy Id mdi trudng dd dugc sir dyng vd cdng bd trudc day bdi Fritz (Fritz vdcs., 1991). Phdn lgp MG dugc thuc hi^n theo phuang phdp ciia Kleven (Kleven, 1998). Cic budc cy the nhu sau: Mau bgnh pham Id phoi cua gd nghi bi bgnh CRD duac cdt nhd \a cho vao 5 ml mdi tmcmg MB . nudi cay trong tu am 37°C \di 5% CO,. Khi mdu mdi trudng nudi cay thay ddi tir mdu dd sang mdu cam hode vdng, ho$c mdl trudng trd nan dye thi mdi trudng nudi cay dugc kiam tra bdng phan ung PCR sir dyng cdp mdi ddc higu MG 14F vd MG 13R cho MG. Nau mdl trudng nuoi cay cho ket qua PCR duong tinh thi MG dugc tiap tyc phdn lgp tren mdi trudng MA \ a nudi cay trong tu am "iT^
vdi 5% CO, trong thdi gian 5-7 ngay.
2.4. Tdch chiet.ADN
ADN cua vi khudn MG dugc tach chiet theo Kit "QIAamp DNA Kit mini preps" cua hUng QIAGEN. Cac budc dugc tian hdnh theo sir chi dan cua Kit, cy tha lOO^iI mau dugc bd sung tham 260^1 dung djch ATL, trdn dau mau va sau dd bd sung tham 30^1 protease K, li 56''C trong 3 gid. Sau khi \x mdu, bd sung tiap 200^1 dung djch AL, trgn dau mdu va sau dd tiap tyc ii mau d 70''C trong 10 phiit. Sau dd Iy tam nhanh vd bd sung 200^1 cdn tuygt doi, trdn dhx mau trong 15 gidy vd Iy tam nhanh cho dich khong cdn bdm tran ndp vd thdnh dng. Tiap theo huyin djch dugc chuyan lan cgt (colunm) (dugc cung cdp kam theo kit), Iy tam 8000 vdng/phiit trong I phlit. Tiap theo, chuyan cdt sang dng tube mdi, bd sung 500^1 dung dich AWI, Iy tam
22
KHOA HQC KVTHUAT THU Y TAP XX S 6 2 - 2013
8000 vdng/phut trong 1 phut. Sau khi Iy tdm, chuyan cgt sang dng tube mdi khdc, bo sung tham 500pl dung dich AW2, ly tam 13500 vdng/
phlit trong 3 phiit. Tiap tuc chuyan cgt sang ong tube mdi, Iy tam vdi tdc dg tdi da ciia mdy trong 1 phut de loai bd hodn todn djch cdn Igi tren cgt.
Chuyen cgt sang dng eppendorf 1,5ml. bd sung 30|JI dgm AE, da nhigt dg phdng I phiit. ly tdm 8000 vdng/phiit trong 1 phiit de thu dung djch ADN. Dung dich DNA dugc bdo qudn d - 2 0 ^ . 2.5. Phan ii-ng PCR
Cdp mdi ddc higu dugc sir dyng trong phan ung PCR cd trinh tg nhu sau: Mdi xudi MGI4F:
5'-GAGCTAATCTGTAAAGTTGGTC-3':
mdi ngugc MG13R: 5pl-GCTTCCTTGCGG TTAGCAAC-3'. Cdp mdi nay dugc thiel ka tran trinh tu gene I6S rRNA ddc hicu cho MG (OIE., 2008). Thdnh phdn phan img PCR gdm cd 2,5 n\ dung dich dem 1 Ox; 1,5nl MgC12 (50 mM); 0,5 pi dNTPs (lOmM); 0,25 ^1 Taq ADN polymerase 5Ln/|il; 14,75 pi nudc; l,5yl mdi xudivd 1,5 plmdi ngugc (15 pmol); 2,5pl ADN sgi khudn. Phdn ung PCR dugc thuc hien theo
chuang trinh; 94"C/5 phiit; 35 chu ky (94"C/1 phiit; 52"C/I phiit; dS^C/l phut); 68"C/I0 phut.
2.6. Dgc ket qua
10|il sdn pham PCR dugc chgy dign di tren gel 1.5% agarose (ABgene. UK) dugc chuan bi trong dung dich dgm lxTAE(10niM Iris, 5mM acetate, 0.1 mM EDTA). KSl qua dign di dugc ghi nhan bdng may dgc GelDoc (BioRad. My).
MarkerADN chudn (DNA MW MARKER, 100 BP LADDER, AMRESCO LLC, USA) dugc chay chung vdi mau de xac dinh kich thudc sdn pham PCR. Mau cho kat qua duang tinh khi sdn pham PCR cd kich thudc Id 185bp.
III. KE I QUA VA THAO LUAN 3.1. Phan lap Mycoplasma
Tir kai qud md kham, nghi ngd bgnh CRD d gd, chiing tdi dd six dyng cdc mdi trudng Mycoplasma Broth (MB) vd Mycoplasma Agar (MA) de phdn lap Mycoplasma. Ket qud phdn lap dugc tha hien d bang 1.
Bdng 1 . Ket qud phan l | p Mycoplasma tir b$nh pham ga nghi m^c CRD
Gidng ga
Ga Ripha G^Ho Ga Ddng lio
Ga M6ng Tdng h0p
Sdm^u kidm tra
23 25 112 14 174
K^t qui nudi cay trdn cac mdi trudng Mycoplasma Broth
Sd miu (+) 12 14 58 5 89
Ty id (%) 52,17 56.00 51,79 35,71 51,15
Mycoplasma Agar Sd m i u (+)
11 10 46 5 72
T } id (%) 47,83 40,00 41,07 35,71 41,38
Ket qua bang 1 cho thdy, cd the phdn lap dugc Mycoplasma d tat cd cdc gidng gd dia phuang nghi mdc CRD. Tren mdi trudng Mycoplasma Broth, ty lg phdn lgp Mycoplasma d bgnh phdm dao ddng tir 35,71% dan 56%. Ty lg phan lap tmng binh tran mdi trudng Mycoplasma Broth Id 51,15%. Tren mdi budng Mycoplasma Agar
ty lg phdn lgp Mycoplasma dao dgng tir 35,71%
den 47,83%. Ty lg phan lgp trung binh tran mdi trudng Mycoplasma Agar Id 41,38%. Kat qud phdn lap Mycoplasma tran mdi trudng ddc MA cho thay khuan Igc dian hinh cua Mycoplasma sau 6 ngdy nudi cay, khuan Igc ciia nd cd dgng tning dp let (binh I).
KHOA HQC KY THUAT THU Y TAP XX S 6 2 - 2013
Hinh 1. KhuS'n l?c Mycoplasma dtidi kfnh hidn vi quang hgc (x40) 3.2. Gidm djnh MG bdng phutrns phap
PCR
3.2.1. Kiim tra dp dgc hifu cua phuong phdp PCR
Cdp moi (primer) ddc higu MGI4F vd MG13R dugc diing trong phdn ung PCR da chdn dodn benh CRD do MG la cdp mdi chuan da dugc cdng bd bdi td chirc OIE. Ddy Id cdp mdi da vd dang dugc su dyng rgng rdi trong cac phdng thi nghiem de chdn doan MG. Cdp mdi ndy dugc thiat ka tran gene I6S rRNA ciia vi khuan MG vk cho ket qua sdn phdm PCR dy kien Id 185 bp. Mac dii cdp mdi dd dugc nghien cuu vd danh gid ve dg ddc hieu vd dg nhgy, nhung khi ung dyng chiing trong chdn dodn MG tgi cdc phdng thi nghigm mdi thi cdp mdi van cdn dugc ddnh gid lai vk do ddc higu. De ddnh gid dg ddc hieu cua phdn ung PCR sir dyng cdp mdi MG14F va MG13R, chung tdi dd sir dyng
2 chiing Mycoplasma ( A K t 19610 vd ATCC 27399) chuan dai dign cho \t i;iilli\cpticum va M svnoviac. Day Id hai lodi Mycoplasma p\m bien gay CRD d gd.Ngodi ra, chiing tdi cdn sir dyng chung virut Lasota vd 3 lodi vi khuan khac Id E.coli, P.multocida vk Salmonella sp lam doi chirng dm.
Ket qua cho thdy chung MG (ATCC 19610) cho ket qud duong tinh khi sir dyng cdp moi ddc higu MGI4F vd MGI3R. Sdn phdm PCR thu dugc cd kich thudc khoang 185 bp (hinh 2), diing vdi cdng bd ciia td chirc OIE (OIE, 2008) khi sir dyng c^p mdi MG14F \ a MGI3R trong phdn umg PCR de chan dodn \IG. Tir hinh 2 cung cho thay phdn ung PCR cho ket qua am tinh ddi vdi MS vd cdc mdu ddi chung am khac.
Nhu vgy, cdp moi MGI4F va MG13R dugc thiat ka tren gen 16S rRNA Id ddc higu cho MG (Hess vdcs., 200"^).
Hinh 2. K^t qud PCR phdt hi$n MG theo qui trinh ciJa OIE, 2008
M Xhirkcr DNA: giing I: Ddi chung duang (MG chiing A TCCI9610): gJng 2. MS chung A TCC 27399: giing3:
virut Niucatxon (Lasota); giing 4: Vi khudn E coli; gieng 5- Vi khudn P.multocida;gieng 6: Vi khudn Salmonella.
Sdn phdm PCR co kich thuoc la 185 bp
3.2.2. Kit qua gidm dfnh MG tir moi trtr&ng phan lgp bdng phtrffng phdp PCR
MG cd tha phdn lap dugc tir dudng hd hdp cua con vat nhu tiii khi, phdi, khi qudn, djch nhdy d cdc xoang, hpng hode tir dudng sinh dye nhu budng trimg, dich hoan. Id huyet cung nhu 24
tir rat nhiau co quan khac. Tuy nhian, mdt trong nhihig trd nggi Idn khi phan lgp MG la do cdm nhidu lodi Mycoplasma khdc nhau cd tha cu tr^
trong dudng hd hdp ciia gia cam, qud trinh phan lap cd tha thu dugc m$t hon hgp gdm nhiau lodi Mycoplasma khdc nhau. Do v§y, sau khi phan
KHOA HOC K? THUAT THU Y TAP XX S 6 2 - 2013
lap cdn phdl cd cac budc kiam tra va gidm dinh lgi bang phdn ijng PCR vdi cdc c?p mdi dac hieu (Ghoraibeh vd Al Roussan, 2008).
Trong nghian cuu ndy, 15 mau Mycoplasma mdi loai trong tdng sd 89 mdu duong tinh vdi Mycoplasma thu dugc Iran mdi trudng phdn lap
MB va 72 mdu duang linh vdi Mycoplasma thu dugc ticn mdi trudng phan lap MA dugc chgn ngau nhien vd dugc giam dinh lai bang phan irng PCR su dyng cdp mdi MG14F vd MG13R ddc higu cho MG. Ket qud dugc the hign d hinh 3 vd bdng 2,
SOObp 400 b p \ 300 b p N ^ 100 b p ^ ^ - 100 ttp l ! ^
M 1 2 3 4 5
Hinh 3. Ket qud giam djnh MG trong moi triTdng ph3n l^p bSng phUdng phdp PCR
M Maikci DNA. gieng I. Doi chung dirong (MG chiing ATCC 19610); giing 2. 3. 4. vd 5 la nhimg mdu duong tinh vm MG. Sdn phdm PCR cd kich thudc Id 185 bp.
Bdng 2. Ket qud giam djnh MG tii mdi tradng phdn l^p bSng phi/dng phdp PCR
Mdi trudng Mycoplasma Broth Mycoplasma Agar
Tdng h^p
Kdt qui PCR S d m l u
Ifiem tra 15 15 30
Sd m i u dUdng tinii
15 15 30
Tyid
(%)
100 100 100Kat qud PCR cho thdy 30/30 (100%) mdu phan lap cho kat qua duang tinh vdi MG. Sdn pham PCR thu dugc cd kich thudc la 185 bp.
diing vdi cdng bo ciia td chiic OIE (OIE, 2008) khi sii dung cap mdi MG14F vd MG13R ddc higu cho MG.
3.2.3. Kit qud chdn dodn MG tir binh pham bdng phtrffng phdp PCR
Trong chdn dodn CRD, PCR ciing dugc cho Id phuang phdp nhanh vd nhgy han phuang
phdp nudi cay phan lap (Kempf va cs., 1993).
Chiing tdi da ung dyng ky thudt PCR trong chan dodn MG tir cdc mdu benh pham Id phdi va khi qudn gd nghi benh. Ket qud dugc trinh bdy tran hinh 4 vdbdng 3.
500 bp 400 b p \ 3 0 0 b p N ^ H i 2 0 0 b p ^ ^ | j 100 bp — ^ ^
Hinh 4. K^t qud phat hi$n MG tif b$nh pham bdng phiidng phdp PCR
M: Marker AND; giing I: doi chirng duang (MG chung ATCC 19610); giing 2, 3. vd 4: cdc mdu duong tinh vdi MG tie binh phdm Id phoi vd khi qudn. Sdn phdm PCR co kich thudc Id 185 bp.
KHOA HQC KY THUAT T H U Y TAP XX S 6 2 - 2013
Theo mgt sd tdi ligu dd cdng bo Irudc dily.
Iy lg phal hign MG bdng phuang phdp PCR tren cdc mdu d|ch ngody hau hgng d gd cho kel qud khdc nhau. Nhu (v lla Lan Id 51,6% (Kempf, 1993). Thd NhT Ky la 29% (Kahya, 2010) vd Ai Cap Id 33,3% (Osman, 2009). Giasuddin (2002) cung dd lirng cho biel ly lg mdc CRD d gia cam lgi Bangladesh Id X.I \%. Trong nghian cuu nay. vdi 25 mau phdi vd 18 mau khi qudn, bang phuang phap PCR dd xac djnh dugc ty lg duong tinh Irung binh dgt 4fi.5"i. Dicu ddng
chii y Id sir dyng bgnh phdm la khi qudn, ty 1^
phdl hign thap ban so vdi bgnh phdm Id phdi (bang 3). Kel qua chdn dodn MG thu dugc trong nghien ciru ndy cd khdc ddi chut so vdi cdc cdng bo d tren. Sg khac bigl nay cd tha la do cdc cdng bo d tren dd sir dyng cdc cap mdi khac nhau trong nghian ciru, cdc cdp mdi khdc nhau thi dg nhgy ciia phan irng PCR Id khdc nhau.
Han n&a cdc yau td ve khi hdu vd dia ly... khac nhau cung dan den ty lg nhiem bgnh CRD tren ddn ga Id khdc nhau.
Bdng 3. Kgt qud c h i n dodn MG tii b$nh p h i m bdng phUOng phdp PCR
Lo9i bdnii phiim
PtidI KhIquSn Tdng h^p
Kdt q u i PCR S d m l u
Iridm tra 25 18 43
S d m d u dudng tinii
12 8 20
T f Id
(%)
48.0 44,4 46,5
Nhu vdy, de phdl hign MG, ngodi phdn img ngung kat nhanh Iran phian kinh, nudi cay phan lap thi phdn ung PCR cd tha dugc dp dyng da chan dodn true tilp tir cdc mdu benh pham Id phdi vd khi qudn ciia gd nghi mdc bgnh. Kel qud ndy hodn todn phy hgp vdi nh&ng nghian ciru trudc day (Feberwee vd cs., 2005; Garcia vd cs., 2005; Kempf vd cs., 1993; Mardassi \a cs., 2005).
IV. K E T L U A N
Ky thugt PCR dd dugc ung dyng thanh cdng trong chdn dodn bgnh CRD do MG gdy ra tren ga ban dja. San phdm PCR thu dugc cd kich thudc la 185 bp, phii hgp vdi cdng bo ciia OIE (OIE, 2008). Kat qud thu dugc tii nghien cuu nay cho thdy ky thuat PCR sir dyng cdp mdi ddc higu MG14F vd MGI3R theo cdng bd cua OIE cd tha dugc su dyng rgng rai trong phdng thi nghigm da chan dodn nhanh vd chinh xdc bgnh CRD do MG gay ra. Trong nghian cuu ndy 20/43 (46,5%) mau bgnh phdm nghi mdc bgnh CRD cho kat qud dugng tinh vdi MG.
TAI LIEU T H A M K H A O 1. Nhii- Vdn Thy (2006). Nghien ciru thiat k^
hg mdi trong Nested PCR da phdt hign mdm bgnh do Mycoplasma gallisepticum tren ga va mgt sd sinh pham thu y, Ludn dn tian si khoa hgc ndng nghigp, Vign thii y. Ha Ngi 2 Feberwee, A.. Mekkes, D.R., de Wit,
J.J.. Hartman, E.G. and Pijpers, A.. 2005.
Comparison of culture, PCR, and different serologic tests for detection of Mycoplasma gallisepticum and Mycoplasma synoviae infections. Avian Dis 49. 260-8.
3. Fritz, B.A., Thomas. C.B.. Van Ess, P. and Yuill, T.M.. 1991. Companson of a modified Edward-type medium and a modified SP4-type medium for primary isolation of Mycoplasma gallisepticum (MG) from chickens vaccinated with the F strain of MG.
Avian Dis 35. 591-8.
4. Garcia, M., Ikuta, N., Levisohn, S. and Kleven, S.H., 2005. Evaluation and comparison of various PCR methods for
26
KHOA HOC KY THUAT THU Y TAP XX SO 2 - 2013
detection of Mycoplasma gallisepticum infection in chickens. Avian Dis 49. 125-32.
5. Ghoraibeh, S. and Al Roussan. D., 2008.
The use of molecular techniques in isolation and characterization of Mycoplasma gallisepticum from commercial chickens in Jordan. Int. J. ofPotilt.Sci 7. 28-35.
6. Hess, M.. Neubauer. C. and Hackl. R., 2007. Interlaboratory comparison of ability to detect nucleic acid of Mycoplasma gallisepticum and MyropUisniu .synoviae by polymerase chain reaction. Avian Pathol 36.
127-33.
1 Kempf, 1. Blanchard, A., Gesberl, F..
Guittei. M and Bennejean, G., 1993. The polymerase chain reaction for Mycoplasma gallisepticum detection. Avian Pathol 22.
739-50.
8. Ley, D.H., 2003, Mycoplasma gallisepticum infeclion In: Diseases of Poultry, llth ed.
Y.M Saif, H J. Barnes. A.M. Fadly, J.R.
Glis-soii, L.R. McDougald, and D.E. Swayne (eds.). Ames. Iowa Slate University Press 122- 144.
9. Mardassi. B.B.. Mohamed. R,B,, Guenri, 1 , Boughallas, S. and Mlik, B , 2005. Duplex PCR to differenlialc between Mycoplasma .synoviae and Mycoplasma gallisepticum on the basis of conserved species-specific sequences of their hemagglutinin genes. J Clin Microbiol 43. 948-58.
lO.OIE.. 2008. Manual of standards for Diagnostic Tests and Vaccines Chapter 2,3.5. Avian mycoplasmosis (Mycoplasma gallisepticum. M. synoviae).
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De dap ijmg sir phat triSn chan nuoi, trong nhihig nam gan day, he thong dich vu ve thu y, chSn nuoi da phat trien mgnh. Tuy nhien, trong dieu kien hi?n nay, cac co quan nha nu'dc vS ch5n nu6i, thu y c6n chira dap img dugc mgt so yeu cau th^c te nhu tuyen truyen, huong dan V.'j thuat va cung cap djch vy de thuc day chan nuoi phat tnen ben viing.
Vi vay, thanh lap Hg! Chan nuoi - Thii y tinh Long An la can thiet nham ho tr? h^ th6ng quan ly nha nuoc, dfing thoi phii hgp voi xu huong xS hpi hoa thu y hi?n nay
Dugc sy cho phep cua UBND tinh Long An (Quy^t dinh sfi 3159/QD-UBND} vk thdnh lap Hgi Chan nuoi-Thu y tinh, Dgi hpi khoa I, nhi|m ky 2013-2018 da dugc tfi chuc long trgng v^o ngay 21/12/2012. D^ii hpi da bau ra Ban ChSp hinh Hfii gfim 15 nguoi, trong d6 ong Dmh Van Thg, Chi c\ic truong Chi eye Thii y dugc bau lam Chii tich, ong Duong Minh Phi va ong Nguyen Van HQu dugc bau \km Pho Chu tich Hpi.
