Detecção do complexo Mycobacterium tuberculosis no leite pela reação em cadeia da polimerase ...
MEDICINA VETERINÁRIA
1023
DETECÇÃO DO COMPLEXO Mycobacterium tuberculosis NO LEITE PELA
REAÇÃO EM CADEIA DA POLIMERASE SEGUIDA DE ANÁLISE DE
RESTRIÇÃO DO FRAGMENTO AMPLIFICADO (PRA)
EDUARDO EUSTÁQUIO DE SOUZA FIGUEIREDO,1 MARLEI GOMES DA SILVA,2 LEILA DE SOUZA FONSECA,3
JOAB TRAJANO SILVA4 E VÂNIA MARGARET FLOSI PASCHOALIN5
06FGRXWRUDQGRGR3URJUDPD&LrQFLDGH$OLPHQWRV8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR(PDLOHGX¿JXHLUHGR#XIUMEU±DXWRUFRUUHVSRQGHQWH
(VSHFLDOLVWDWpFQLFRGHODERUDWyULR8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR
'6FSURIHVVRUWLWXODU8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR
'6FSURIHVVRUDGMXQWR,8QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR
'6FSURIHVVRUDGMXQWR,98QLYHUVLGDGH)HGHUDOGR5LRGH-DQHLUR
RESUMO
Mycobacterium bovis p PHPEUR GR FRPSOH[R
Mycobacterium tuberculosis (MTBC), grupo este composto por espécies com grande homologia genética. É o
DJHQWHHWLROyJLFRGDWXEHUFXORVHERYLQDLPSRUWDQWH]RRnose transmissível ao homem, principalmente através da
LQDODomRGREDFLORHRXSHORFRQVXPRGHOHLWHHGHULYDGRV
QmRSDVWHXUL]DGRV SURYHQLHQWHV GH YDFDV WXEHUFXORVDV
2REMHWLYRGHVWHHVWXGRIRLSDGURQL]DUDLGHQWL¿FDomRGH
PLFREDFWpULDVGRFRPSOH[RM. tuberculosis presentes no
OHLWHSRUPHWRGRORJLDPROHFXODU)H]VHDH[WUDomRGH'1$
GLUHWDPHQWHGROHLWHFRQWDPLQDGRHUHDOL]RXVHDLGHQWL¿FDção molecular pela reação em cadeia da polimerase seguida
GHDQiOLVHGHUHVWULomRGRIUDJPHQWRDPSOL¿FDGR35$
8WLOL]DUDPVHLQKDJHQVGHUHIHUrQFLDHOHLWHFUXDUWL¿FLDOmente contaminado com M. bovis ,3 8P IUDJPHQWR GH
SEGRJHQHhspIRLDPSOL¿FDGRWUDWDGRFRPBstEII
e Hae,,, H HPSUHJRXVH R SHU¿O GH UHVWULomR HQ]LPiWLFD
REWLGR SDUD LGHQWL¿FDU R FRPSOH[R M. tuberculosis no
OHLWH&RPD35$IRLSRVVtYHOGHWHFWDUFRPHVSHFL¿FLGDGH
HVHQVLELOLGDGHDSUHVHQoDGHM. bovisHPDWp8)&P/
GH OHLWH$ PHWRGRORJLD SDGURQL]DGD SRGHUi DX[LOLDU RV
PpWRGRVPLFURELROyJLFRVHELRTXtPLFRVWUDGLFLRQDOPHQWH
XVDGRVQDLGHQWL¿FDomRGREDFLORHPDOLPHQWRVVXVSHLWRV
de contaminação, como, por exemplo, o leite proveniente
GHDQLPDLVVXVSHLWRVGHLQIHFomRSRUM. bovis.
3$/$95$6&+$9(6 $QiOLVHGHSHU¿OGHUHVWULomRHQ]LPiWLFD35$FRPSOH[RMycobacterium tuberculosis, leite,
Mycobacterium bovis, limite de detecção (PCR).
ABSTRACT
DETECTION OF Mycobacterium tuberculosis&203/(;%<3&55(675,&7,21)5$*0(17/(1*7+
32/<025),60$1$/<6,62)7+(+63*(1(
Mycobacterium bovisLVDPHPEHURIWKHM. tuberculosis FRPSOH[DJURXSFRPSRVHGE\VSHFLHVZLWKKLJK
JHQHWLFKRPRORJ\7KHSDWKRJHQLVWKHHWLRORJLFDODJHQWRI
ERYLQHWXEHUFXORVLVDQLPSRUWDQW]RRQRVLVWKDWLVPDLQO\
WUDQVPLWWHGE\LQKDODWLRQRILQIHFWLRXVGURSOHWQXFOHLRUE\
LQJHVWLRQRIPLONDQGFUXGHPLONGHULYDWLYHSURGXFWVIURP
WXEHUFXORVLVFRZV7KHGH¿QLWLYHLGHQWL¿FDWLRQRIM. bovis,
XSWRVSHFLHVOHYHOLVWLPHFRQVXPLQJDQGGLI¿FXOW,QWKLV
ZRUNWKHREMHFWLYHZDVWRVWDQGDUGL]HDSRO\PHUDVHFKDLQ
UHDFWLRQIROORZHGE\DQHQ]\PHUHVWULFWLRQDQDO\VLVLQRUGHU
WRLGHQWLI\WKHM. tuberculosis FRPSOH[LQPLONZLWKRXWD
PLFURELRORJLFDO LVRODWLRQ VWHS 5HIHUHQFH VWUDLQV DQG UDZ
PLONVHHGHGZLWKM. Bovis, ZHUHXVHGDVWKHVWDUWLQJPDWHrial. $SEIUDJPHQWRIWKHhspJHQHZDVDPSOL¿HG
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
1024
FIGUEIREDO, E. E. de S. et al.
DQGGLJHVWHGE\WZRUHVWULFWLRQHQ]\PHVBstEII and HaeIII.
7KHREWDLQHGSUR¿OHZDVXVHGWRLGHQWLI\WKHM. tuberculosis
complex LQPLON7KHPLQLPXPOLPLWRIGHWHFWLRQRIM. bovis
LQPLONZDV&)8P/35$PHWKRGRORJ\SURYHGWREH
DVSHFL¿FDQGVHQVLEOHPHWKRG,WFDQEHXVHGWRDVVLVWWKH
PLFURELRORJLFDODQGELRFKHPLFDOPHWKRGVFRPPRQO\XVHG
WRLGHQWLI\LQJWKHEDFLOOLLQFOLQLFDOVDPSOHVDVPLON
KEY WORD: Detection limit (PRA), Mycobacterium tuberculosis complex, PLON Mycobacterium bovis, Restriction Enzyme Analysis (PCR),
INTRODUÇÃO
O Complexo Mycobacterium tuberculosis
(MTBC) é composto pelas espécies Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Bacille Calmette-Guérin
(BCG), Mycobacterium microti, Mycobacterium
canetti, Mycobacterium caprae e Mycobacterium
pinnipedii. O M. tuberculosis e o M. bovis se
destacam por serem os agentes causadores da
WXEHUFXORVHKXPDQDHERYLQDUHVSHFWLYDPHQWH$
FHSDLQIHFWDQWHERYLQDpDPDLVSHULJRVDGHQWUHDV
HVSpFLHVGHVWHFRPSOH[RVHQGRFDSD]GHLQIHFWDU
PXLWDVHVSpFLHVGHPDPtIHURVLQFOXLQGRRKRPHP
(O’ REILLY & DABORN, 1995).
$WXEHUFXORVHERYLQDpXPD]RRQRVHGRHQoDRXLQIHFomRQDWXUDOPHQWHWUDQVPLVVtYHOHQWUH
RVDQLPDLVYHUWHEUDGRVHRKRPHPWUDQVPLVVtYHO
GRJDGRSDUDKXPDQRVGLUHWDPHQWHSHODYLDDHUygena, mediante a inalação do M. bovis suspenso
no ar, e indiretamente, pelo consumo de leite e de
SURGXWRV OiFWHRV QmR IHUYLGRV RX SDVWHXUL]DGRV
HFRPPHQRUIUHTrQFLDSHODLQJHVWmRGHFDUQH
HSURGXWRVFiUQHRVFRQWDPLQDGRV:+2
GRANGE & YATES, 1994).
2FRQWUROHGDWXEHUFXORVHERYLQDHRVSURJUDPDVGHHUUDGLFDomREDVHDGRVQRWHVWHGHWXEHUFXOLQDHVDFULItFLRGRVDQLPDLVSRVLWLYRVMXQWR
FRPDSDVWHXUL]DomRGROHLWHGLPLQXtUDPGUDVWLFDPHQWHDLQFLGrQFLDGHWXEHUFXORVHERYLQDQRV
DQLPDLVHQRVKXPDQRVQRVSDtVHVLQGXVWULDOL]DGRV(PSDtVHVRQGHDSUHYDOrQFLDGDWXEHUFXORVH
ERYLQDpHOHYDGDRXRQGHRVSURJUDPDVGHFRQWUROH
HHUUDGLFDomRQmRH[LVWHPRXDLQGDHVWmRHPIDVH
de implantação, o leite e seus derivados não pasWHXUL]DGRVVmRFRQVLGHUDGRVXPDVGDVSULQFLSDLV
IRQWHV GH WUDQVPLVVmR GD GRHQoD $6+)25'
2001). Em tais países, o leite contaminado com
M. bovis, oriundo de vacas doentes, é a principal
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
FDXVDGHOLQIRDGHQRSDWLDVFHUYLFDLVHVFUyIXODH
GH RXWUDV IRUPDV GD WXEHUFXORVH H[WUDSXOPRQDU
HPKXPDQRV02'$HWDO&286,9,HW
al., 1998).
2WHVWHLPXQROyJLFRLQGLUHWRGHWXEHUFXOLQL]DomRpRPpWRGRGLDJQyVWLFRSDGUmRGHHVFROKD
SDUD LGHQWL¿FDU RV DQLPDLV WXEHUFXORVRV 2,(
2000) e consiste na avaliação de uma reação de
KLSHUVHQVLELOLGDGH WDUGLD GHÀDJUDGD HP DQLPDLV
SUHYLDPHQWH H[SRVWRV DR EDFLOR GD WXEHUFXORVH
SURYRFDGDHPUHVSRVWDjLQMHomRLQWUDGpUPLFDGH
WXEHUFXOLQDDYDOLDGRDSyVKRUDV$VHQVLELOLGDGHGRWHVWHGHWXEHUFXOLQL]DomRYDULDHQWUH
HQTXDQWR VXD HVSHFL¿FLGDGH IRL HVWLPDGD
HQWUH$VHQVLELOLGDGHSRGHVHUDIHWDGD
SRUIDWRUHVLDWURJrQLFRV&255($&255($
WDLVFRPRGHVHQVLELOL]DomRLPXQRVXSUHVVmRSyVSDUWRLQWHUYDORSyVLQIHFomR&255($
& CORREA, 1981), assim como por variação da
GRVHHSRWrQFLDGDWXEHUFXOLQDXWLOL]DGDHGDYDULDELOLGDGHQDDYDOLDomRGRHGHPDSHORREVHUYDGRU
$HVSHFL¿FLGDGHSRUVXDYH]pLQÀXHQFLDGDSHOD
VHQVLELOL]DomRTXHSRGHRFRUUHUSHODH[SRVLomRGR
animal a M. avium, M. paratuberculosis ou por
PLFREDFWpULDGRPHLRDPELHQWH2¶5(,//<HWDO
021$*+$1 HW DO (VVDV IDOKDV
QRGLDJQyVWLFRGHWXEHUFXORVHERYLQDSURYRFDP
UHVXOWDGRVIDOVRVSRVLWLYRVDQLPDLVVHPGRHQoD
H IDOVRVQHJDWLYRV DQLPDLV WXEHUFXORVRV FRP
UHVXOWDGRGDSURYDQHJDWLYR2GLDJQyVWLFRFRQ¿UPDWyULRpIHLWRDSyVDPRUWHGRDQLPDOHUHTXHU
R LVRODPHQWR H D LGHQWL¿FDomR GH M. bovis, um
SURFHGLPHQWRWUDEDOKRVRHGHPRUDGRTXHSRGH
OHYDUDWpGR]HVHPDQDV&251(5
1R %UDVLO RV tQGLFHV R¿FLDLV GH WXEHUFXORVHERYLQDVmRGHGRUHEDQKRQDFLRQDO
LQIHFWDGRRTXHUHSUHVHQWDFHUFDGHPLOK}HV
GHDQLPDLVWXEHUFXORVRV3HVTXLVDVUHFHQWHVFRQ¿UPDPTXHDLQIHFomRVHFRQFHQWUDHPERYLQRV
Detecção do complexo Mycobacterium tuberculosis no leite pela reação em cadeia da polimerase ...
OHLWHLURVPDVSULQFLSDOPHQWHQDTXHOHVUHEDQKRV
FRPDOJXPJUDXGHWHFQL¿FDomRHPTXHDVWD[DV
GH LQIHFomR SRGHP FKHJDU D GH UHEDQKRV
FRPSHORPHQRVXPDQLPDOLQIHFWDGRBRASIL,
0HVPR QHVVHV UHEDQKRV WHFQL¿FDGRV R
OHLWH PXLWDV YH]HV p GLVWULEXtGR in natura para
RFRQVXPRKXPDQR(VWLPDVHTXHD
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WUDQVPLVVmRGHWXEHUFXORVHEHPFRPRGHRXWUDV
HQIHUPLGDGHV DR KRPHP DWUDYpV GR FRQVXPR
GHOHLWHHGHULYDGRVFUXV),*8(,5('2
58**,(52
$SHVDUGHQmRKDYHUHVWDWtVWLFDVTXHTXDQWL¿TXHPDVSHUGDVHFRQ{PLFDVLPSRVWDVSHODWXEHUFXORVHERYLQDQR%UDVLOWDPSRXFRRFRPSURPHWLmento humano, o Ministério da Agricultura lançou
HPMDQHLURGHVXDFDPSDQKDGHFRPEDWHD
HVVDHQIHUPLGDGH23URJUDPD1DFLRQDOGH&RQWUROH H (UUDGLFDomR GH %UXFHORVH H7XEHUFXORVH
31&(%7YLVDFRPEDWHUHVVDVHQIHUPLGDGHVQD
SRSXODomRERYLQDHEXEDOLQDGLPLQXLQGRDLQFLGrQFLDHSUHYDOrQFLDGHVWDVD¿PGHPLQLPL]DU
DVSHUGDVHFRQ{PLFDVHRIHUHFHUJDUDQWLDVGHLQRcuidade dos alimentos, considerando tanto a carne
como o leite e derivados, ao consumo interno, e
aumentar a competitividade dos nossos produtos
no mercado internacional.
6HJXQGR6,1+$GDVYDFDV
HPODFWDomRTXHVHDSUHVHQWDPSRVLWLYDVDRWHVWH
GHWXEHUFXOLQL]DomRHOLPLQDPREDFLORQROHLWH
PDVDSHQDVGHVVHVDQLPDLVHOLPLQDPM. bovis
HPTXDQWLGDGHFDSD]GHVHUPHQVXUDGDSRUFXOWXUD3RURXWURODGR6,1+$H=$1,1,HW
DOGHPRQVWUDUDPTXHYDFDVFRPLQIHFomR
FOtQLFDHVXEFOtQLFDSRGHPH[FUHWDUGH[2 a
105XQLGDGHVIRUPDGRUDVGHFRO{QLDV8)&GHM.
bovis por mL de leite. Além disso, o leite pode ser
FRQWDPLQDGRSRUEDFLORVH[yJHQRVSURYHQLHQWHV
GHHTXLSDPHQWRVGHRUGHQKDVXMRVHPDOODYDGRV
$%5$+27UDWDVHGHGLVFUHSkQFLDTXH
GUDPDWL]DDQHFHVVLGDGHGHVHREWHUXPPpWRGR
UiSLGRVHQVtYHOHVSHFt¿FRHVHJXURSDUDLGHQWL¿FDUREDFLORERYLQRQROHLWH
A Polimerase chain reaction Restriction enzyme Analysis 35$pXPDIHUUDPHQWDPROHFXODU
TXHMXQWDPHQWHFRPD3&5RVHTHQFLDPHQWRGH
1025
'1$3$,HWDO0&1$%%HWDO
RVPpWRGRVGHKLEULGL]DomRFRPVRQGDVGH'1$
(//1(5HWDODVDQiOLVHVGHSHU¿VGH
iFLGRVPLFyOLFRV/(,7(HWDODWpFQLFD
de microarrays)8.86+,0$HWDO., 2003), vêm
VHQGRXWLOL]DGRVSDUDLGHQWL¿FDUGHPDQHLUDPDLV
SUHFLVD H UiSLGD DV HVSpFLHV GH PLFREDFWpULDV
previamente isoladas.
9iULRV PpWRGRV EDVHDGRV HP 3&5 SDUD
LGHQWL¿FDomRGHM. bovis:$5'6HWDO
=$1,1, HW DO H &2//,16 HW DO
65((9$76$1HWDO7$</25HW
DO=$1,1,HWDO=80$55È*$
HWDODQG3(5(=HWDOMiIRUDPGHVFULWRV
EDVHDGRVQDDPSOL¿FDomRGHGLIHUHQWHVJHQHVWDLV
FRPRSQF$%$5281,HWDO TRIGUI et
DOgyrB (LEÃO et al., 2005), oxyR (SRE(9$76$1HWDO 1996) e katG+$$6 H
QDVHTrQFLDRvD1Rv2031c 52'5,*8(=HODO
1995), a região IS1081TXHWHPFLQFRRXVHLVFRpias em M. bovis (WARDS et al.,=$1,1,
et al., 1998), o gene aspb70 (COLLINS et al.,
1991 e WARDS et al., 1995), IS6110 (NOLTE et
DO3(5(=HWDO7$</25HWDO
=$1,1,HWDO=80$55È=$HWDO
2001 e 2005) e o gene hsp65 65((9$76$1HW
DO=80$55$*$HWDO
Entretanto, para evitar a presença de possíYHLVLQLELGRUHVSUHVHQWHVQDDPRVWUDFOtQLFDTXH
SRGHULDPLQWHUIHULUFRPDSHUIRUPDQFHGD3&5
'(:,7HWDO&/$55,'*(HWDO
12/7( HW DO /,e%$1$ HW DO 0$1*,$3$1 HW DO RV SHVTXLVDGRUHV
LQFOXHPRLVRODPHQWRSUpYLRGDVPLFREDFWpULDV
SDUD Vy HQWmR SURFHGHU j H[WUDomR GR '1$ H j
3&5(VVHSURFHGLPHQWRDXPHQWDDVHQVLELOLGDGH
GRPpWRGRPDVUHTXHUTXHVHMDIHLWRXPWUDWDmento inicial de descontaminação da amostra com
1D2+ FRQFHQWUDGR PpWRGR GH 3HWURII VHQGR
TXHFHUFDGHGDVDPRVWUDVFOtQLFDVWUDWDGDV
GHVVDPDQHLUDQmRDSUHVHQWDPFRO{QLDVYLiYHLV
$SHVDU GD YDVWD XWLOL]DomR GD 35$ SDUD
LGHQWL¿FDUHFDUDFWHUL]DUQRYDVHVSpFLHVGHPLFREDFWpULDVSRXFRVHVWXGRVYrPVHQGRUHDOL]DGRV
SDUDDQDOLVDUDH¿FLrQFLDGD35$QDLGHQWL¿FDção do complexo Mycobacterium turberculosis
em leite e tecidos. No Brasil, ARAÚJO, et al.
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
1026
XVDUDPD35$SDUDLGHQWL¿FDUDSUHVHQoD
do complexo Mycobacterium tuberculosis em
TXDWURFRO{QLDVLVRODGDVGHFDUFDoDVERYLQDVFRP
OHV}HV GH WXEHUFXORVHQmRLGHQWL¿FDGRVQD3&5
HVSpFLHHVSHFt¿FD SDUD M. bovis e os isolados
IRUDP LGHQWL¿FDGRV FRPR SHUWHQFHQWHV DR FRPplexo Mycobacterium tuberculosisFRQ¿UPDQGR
DVXVSHLWDGHWXEHUFXORVH8PHVWXGRVHPHOKDQWH
IRLUHDOL]DGRSRU.218.HWDOTXHLGHQWL¿FDUDPSRU35$GLYHUVDVPLFREDFWpULDVDWtSLFDV
LVRODGDVGHOHLWHFUXQD7XUTXLDGHPRQVWUDQGRD
FRQ¿DELOLGDGHGHVVDWpFQLFDPROHFXODUHPFRPSDUDomRFRPDLGHQWL¿FDomRELRTXtPLFD
Neste estudo, empregou-se a PRA para analisar e detectar a presença de M. bovis em amostras de
OHLWHLGHQWL¿FDQGRRDWUDYpVGRSHU¿OGHUHVWULomR
do complexo M. tuberculosis2PpWRGRXWLOL]DGR
EDVHLDVH QD REWHQomR GH DPRVWUDV GH '1$ GH
PLFREDFWpULDVGLUHWDPHQWHGROHLWHVHPHWDSDVGH
LVRODPHQWRVHJXLGDGDDPSOL¿FDomRGHSDUWHGDVHTrQFLDGRJHQHhsp65 (altamente conservada entre
o gênero 0\FREDFWHULXP ) por PCR, e da digestão
GRIUDJPHQWRDPSOL¿FDGRFRPDVHQ]LPDVGHUHVtrição BstEII e HaeIII (TELENTI et al., 1993).
FIGUEIREDO, E. E. de S. et al.
3UHSDUDomRGRLQyFXORGHM. bovis
Uma alçada contendo células de M. bovis IP
HPIDVHDWLYDGHFUHVFLPHQWRIRLUHVVXVSHQVDHP
P/GHiJXDGHVWLODGDHVWpULOHPWXERIDOFRQGH
P/FRQWHQGRSHUyODVGHYLGUR$SyVDJLWDomR
YLJRURVDDMXVWRXVHDWXUELGH]GDVXVSHQVmRD
pela escala McFarland, correspondendo a 3,0 x 108
8)&P/&286,16HWDO.2*$*2=HW
DO3DUDFRQIHULUDFRQFHQWUDomRGDVFpOXODV
QDVXVSHQVmRHVWRTXHGLOXLo}HVVHULDGDVGH
IRUDPSUHSDUDGDVHLQRFXORXVHP/GHFDGD
GLOXLomRHP/-3VHQGRLQFXEDGRVD&
SRUDWpRLWRVHPDQDV(PSUHJRXVHRQ~PHURGH
FRO{QLDV8)&REWLGDVSDUDDMXVWDURQ~PHURGH
FpOXODVQDVXVSHQVmRHVWRTXH'LOXLo}HVVHULDGDV
de 100 a 108 &)8IRUDPSUHSDUDGDVFHQWULIXJDGDV
e o sedimento inoculado em 1mL de leite para
posterior análise.
Extração de DNA de culturas puras
2EWHYHVH'1$JHQ{PLFRGHFDGDXPDGDV
linhagens de Mycobacterium pelo método da lise
WpUPLFD&pOXODVFUHVFLGDVHPPHLR/3RX/3-IRram ressuspensas em 400µL tampão TET (10mM
MATERIAL E MÉTODOS
7ULV+&O P0 ('7$ FRP 7ULWRQ ;
S+ 6XEPHWHXVH D VXVSHQVmR FHOXODU D WUrV
0LFURRUJDQLVPRVXWLOL]DGRVHPHLRVGHFXOWXUD
FLFORVGHFKRTXHWpUPLFRGH]PLQXWRVD&
8WLOL]DUDPVH WUrV OLQKDJHQV GH UHIHUrQFLD VHJXLGRVSRUGH]PLQXWRVD&FHQWULIXJDGRV
SDUDSDGURQL]DUDPHWRGRORJLDM. bovis (Instituto D[JSRUFLQFRPLQXWRVHXWLOL]DUDPVH/
Pasteur - IP, Paris) representando o complexo M. GR VREUHQDGDQWH FRPR PROGH GH '1$ QD 3&5
tuberculosis, M. aviumVRURWLSR$7&& 6,/9$HWDO
e M. fortuitum ATCC 6841. Mantiveram-se as
FRO{QLDVGHM. avium e de M. fortuitum em meio Extração do DNA de M. bovis HPOHLWHDUWL¿FLDOGHFXOWXUD/RZHQVWHLQ-HQVHQ/-HQTXDQWRTXH mente contaminado
a de M. bovis,HPPHLR/-FRPSLUXYDWRGHVyGLR
&RQWDPLQDUDPVHDUWL¿FLDOPHQWHDPRVWUDV
(LJP).
de leite de vacas sadias (1 mL) com M. bovis IP
FRPR GHVFULWR DQWHULRUPHQWH VHQGR XWLOL]DGDV
2EWHQomRGDVDPRVWUDVGHOHLWH
SDUDDREWHQomRGHPROGHVGH'1$SDUDDVUHDo}HV
Escolheram-se vacas negativas ao teste de GH3&5$H[WUDomRGR'1$IRLIHLWDGHDFRUGR
WXEHUFXOLQL]DomRQmRWXEHUFXORVDVSDUDFROHWDGH FRPPRGL¿FDomRGRPpWRGRGHVFULWRSRU/(2
DPRVWUDVGHOHLWHSDUDDSDGURQL]DomRGRPpWRGR HW DO 5HVXPLGDPHQWH FHQWULIXJDUDPVH
$SyVODYDJHPGR~EHUHGRVDQLPDLVIRUDPFROH- amostras (1mL) previamente contaminadas a
tados, assepticamente, cerca de 500 mL de leite, 10.000xg por cinco minutos, sendo descartado
por ordenha manual, descartando-se os primeiros R VREUHQDGDQWH REWLGR GH FDGD XPD GHODV H R
MDWRV
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
Detecção do complexo Mycobacterium tuberculosis no leite pela reação em cadeia da polimerase ...
VHGLPHQWR ODYDGR WUrV YH]HV FRP WDPSmR 7(
P07ULV+&OP0('7$S+UHVVXVSHQVRHP/GH7(H/GHVROXomRGH
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/GH6'6H7(IRUDPDGLFLRQDGRVDWp
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DFHWDWRGHVyGLR0S+VHQGR/GH
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FHQWULIXJDomRD[JSRUTXLQ]HPLQXWRV
2'1$SUHFLSLWDGRIRLODYDGRFRP/GH
HWDQRODHUHVVXVSHQVRHP/GHiJXD
miliQ estéril. Empregaram-se 5 µL da suspensão
na reação da PCR.
PCR – Restriction Enzyme Analysis (PRA)
A mistura de reação para PCR continha
5µL do molde de DNA, 50 pmol de oligonuJOHRWtGHRV SXUL¿FDGRV H GHVVDOLQL]DGRV 7E
¶$&&$$&*$7**7*7*7&&$7 H7E
(5’- CTTGTCGAACCGCATACCCT) (Invitrogen) (TELENTI et al., 1993), 2,0mM MgCl2,
P0GHFDGDGHR[LUULERQXFOHRVtGHRWULIRVIDWR
G173V3KDUPDFLD%LRWHFKH8GH7DT'1$
SROLPHUDVHUHFRPELQDQWH,QYLWURJHP/LIH7HFKQRORJLHV/GRWDPSmRGDHQ]LPD[FRQFHQWUDGRS+HPXPYROXPH¿QDOGH/
Procedeu-se à PCR em termociclador automático
3HUNLQ(OPHU*HQH$PS3&5V\VWHPQDV
VHJXLQWHVFRQGLo}HV&SRUGH]PLQXWRV
FLFORVGHXPPLQXWRD&GHVQDWXUDomRXP
PLQXWRD&DQHODPHQWRXPPLQXWRD&
(extensão), e mais um ciclo de sete minutos a
&SDUDH[WHQVmR¿QDOGRIUDJPHQWR2VSURGXWRVGHSEDPSOL¿FDGRVIRUDPDQDOLVDGRV
SRUHOHWURIRUHVHHPJHOGHDJDURVHFRUDGRV
FRPEURPHWRGHHWtGLRJP/HYLVXDOL]DGRV
HPWUDQVOXPLQDGRUGH89QP
,QFXEDUDPVHTXLQ]HPLFUROLWURVGRSURGXWRGHUHDomRGH3&5FRP8GDHQ]LPDBstEII
%LRDJHQF\D&SRUGXDVKRUDVHRXWURV/
GRSURGXWRFRPDHQ]LPDHae,,,%LRDJHQF\D
&WDPEpPSRUGXDVKRUDV
,GHQWL¿FDomRGR&RPSOH[RM. tuberculosis
5HDOL]RXVHDDQiOLVHGDUHVWULomRHQ]LPiWLFD
HPJHOGHDJDURVHHFRPDX[tOLRGRSDGUmRGH
IUDJPHQWRVGHSE3520(*$FRPSDUDUDP
se os resultados com dendogramas disponíveis
QD OLWHUDWXUD '(9$//2,6 HW DO /(2
et al., 2004).
RESULTADOS
,GHQWL¿FDomR GR FRPSOH[R M. tuberculosis pela
PRA
3DUDLGHQWL¿FDURFRPSOH[RM. tuberculosis
07%&IRUDPUHDOL]DGDV3&5VXVDQGRRV'1$V
PROGHVREWLGRVGDVOLQKDJHQVGHUHIHUrQFLDFRPRR
M. bovis IP (controle positivo), M. avium sorotipo
$7&&e M. fortuitum ATCC 6841 (controles negativos), e os primers TB11 e TB12 para
DPSOL¿FDURJHQHhsp65VHQGRTXHRIUDJPHQWR
DPSOL¿FDGRIRLGLJHULGRFRPGXDVHQGRQXFOHDVHV
BstEII e HaeIII.
&RPR SRGH VHU REVHUYDGR QD )LJXUD $
UDLDVHRFRUUHXDPSOL¿FDomRGHXPIUDJPHQWRGHESTXDQGRR'1$GDVWUrVHVSpFLHV
de MycobacteriumIRLXWLOL]DGRFRPRPROGHQD
reação de PCR.
$FOLYDJHPGRIUDJPHQWRGHSEGRJHQH
hsp65 com as endonucleases BstEII e HaeIII amSOL¿FDGRGRFRPSOH[RM. tuberculosis deveria geUDUGRLVFRQMXQWRVGHIUDJPHQWRVSESE
SE H SE SE SE UHVSHFWLYDPHQWH
'(9$//2,6HWDO/(2HWDO
'H IDWR TXDQGR R IUDJPHQWR GH SE
DPSOL¿FDGRDSDUWLUGR'1$PROGHGH M. bovis
IRLWUDWDGR com as endonucleases BstEII e HaeIII
)LJXUD%H&UDLDVREWLYHUDPVHRVSHU¿VGH
restrição esperados para o complexo M. tuberculosisSESESEHSESESE
respectivamente, correspondentes ao complexo
M. tuberculosis4XDQGRRVDPSOL¿FDGRVREWLGRV
do DNA de M. avium (Figuras 1B e 1C, raias 1) e
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
1028
FIGUEIREDO, E. E. de S. et al.
de M. fortuitum)LJXUDV%H&UDLDVIRUDP SESEHSEFRPRHVSHUDGRSDUD
WUDWDGRVFRPDVHQGRQXFOHDVHVREWLYHUDPVHGRLV HVVDV GXDV HVSpFLHV '(9$//2,6 HW DO FRQMXQWRV GH IUDJPHQWRV SE SE H GH LEÃO et al., 2004).
SEHSEHSESEHSEHGH
FIGURA 1.,GHQWL¿FDomRGRFRPplexo M. tuberculosis por PRA:
3$,1(/$ ± '1$ REWLGR GH WUrV
GLIHUHQWHV Mycobacterium sp. por
OLVH WpUPLFD DPSOL¿FDGR FRP RV
ROLJRQXOHRWtGHRV 7% H 7%
IUDJPHQWRVDPSOL¿FDGRVHYLVXDOL]DGRVHPJHOGHDJDURVHFRUDGR
FRP EURPHWR GH HWtGHR OLQKD 3
SDGUmR GH WDPDQKR GH IUDJPHQWR
SE 3520(*$ OLQKD M.
avium VRURWLSR $7&& linha 2, M. fortuitum$7&&
linha 3, M. bovis,33$,1(/%±
$QiOLVHGRVIUDJPHQWRVGHGLJHVWmR
HQ]LPiWLFDSRUHOHWURIRUHVHHPJHO
GHDJDURVHGRVDPSOLFDGRVREWLGRVQRSDLQHO$OLQKD3SDGUmRGHWDPDQKRGHIUDJPHQWRVSEOLQKDVGHDSHU¿OGHEDQGDVDSyVDGLJHVWmRFRP
DHQ]LPDBstEII para M. avium, M. fortuitum e M. bovisUHVSHFWLYDPHQWH3$,1(/&±2SHU¿OGHEDQGDVREWLGRVDSyV
GLJHVWmRFRPDHQ]LPDHaeIII para os mesmos microrganismos.
Detecção pela PRA de M. bovis HPOHLWHDUWL¿FLDOmente contaminado
3DUD YHUL¿FDU VH VHULD SRVVtYHOLGHQWL¿FDU
SRU35$PLFREDFWpULDVGRFRPSOH[RM. tuberculosisTXHHYHQWXDOPHQWHVHULDPH[FUHWDGDVQR
OHLWH GH YDFDV WXEHUFXORVDV SURFHGHXVH D XPD
adaptação da metodologia, como descrito antes,
FRQWDPLQDQGRVHDUWL¿FLDOPHQWHDPRVWUDVGHOHLWH
cru com M. bovis,3HPGLIHUHQWHVFRQFHQWUDo}HV
(100±88)&P/GHOHLWH
Preparou-se o DNA a partir de 1mL das
amostras de leite contaminado, mediante método
GH H[WUDomR FRP OLVR]LPD6'6 DQWHULRUPHQWH
XWLOL]DGR FRPR PROGH GH '1$ QDV UHDo}HV GH
3&5 SDUD DPSOL¿FDomR GD VHTrQFLD GR JHQH
hsp652SURGXWRDPSOL¿FDGRIRLWUDWDGRFRPDV
endonucleases BstEII e Hae,,,HRVSHU¿VGHUHVWULomRIRUDPDQDOLVDGRVHPJHOGHDJDURVH
&RPRPRVWUDGRQD)LJXUD$IRLSRVVtYHO
REWHU'1$DPSOL¿FiYHOGHPLFREDFWpULDVSUHVHQCiência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
tes no leite sem a necessidade de isolar tais microrJDQLVPRV&RPRHVSHUDGRTXDQGRRDPSOL¿FDGR
GHESIRLGLJHULGRFRPDVHQGRQXFOHDVHVBstEII
e HaeIII (Figura 2B raias: a - h e i - oREWHYHVH
RSHU¿OGHIUDJPHQWRVGHUHVWULomRSESE
SESESESEFRUUHVSRQGHQWHDM.
bovis$OpP GLVVR HVWDEHOHFHXVH TXH R OLPLWH
mínimo de detecção de M. bovis com essa meWRGRORJLDIRLGH 8)&P/GHOHLWH)LJXUD$
raia 8).
$35$RWLPL]DGDQHVWHHVWXGRpXPPpWRGR
HVSHFt¿FRVHQVtYHOHUiSLGRTXHSHUPLWLXGHWHFWDU
PHPEURVGRFRPSOH[RM. tuberculosis no leite,
VHPDQHFHVVLGDGHGHID]HURLVRODPHQWRGHPLcroorganismos.
7DQWRDH[WUDomRGH'1$GHPLFREDFWpLUDV
GLUHWDPHQWHGROHLWHTXDQWRDDPSOL¿FDomRGHVVHV
PROGHVGH'1$IRUDPEHPVXFHGLGRV2PpWRGR
XWLOL]DGRQHVWHHVWXGRSDUDLVRODU'1$GDVPLFREDFWpULDVFRQWDPLQDQWHVGROHLWHSHUPLWLXH[WUDLU
R'1$GHPLFREDFWpULDVPHVPRQDSUHVHQoDGH
Detecção do complexo Mycobacterium tuberculosis no leite pela reação em cadeia da polimerase ...
1029
PLQHUDLVSURWHtQDVJOyEXORVGHJRUGXUDHFpOXODV YHOGHH[WUDomRRXDLQGDLQLELUDDPSOL¿FDomRGR
HSLWHOLDLVGRKRVSHGHLURSUHVHQWHVQROHLWHRVTXDLV '1$PROGHSRULQLELomRGD'1$SROLPHUDVH
SRGHULDPUHGX]LUDTXDQWLGDGHGH'1$WRWDOSDVVt-
FIGURA 2. Limite de
detecção de M. bovis em
OHLWHDUWL¿FLDOPHQWHFRQtaminado. 3$,1(/$±
P/GHOHLWHFUXDUWL¿FLDOmente contaminado com
GLOXLo}HVGHM. bovis 108
a 10 0 8)&P/ '1$
extraído pelo método de
OLVR]LPD6'6DPSOL¿FDGRHYLVXDOL]DGRHPJHO
GH DJDURVH FRUDGR
FRP EURPHWR GH HWtGHR
/LQKD3SDGUmRGHWDPDQKRGHIUDJPHQWRSE3520(*$OLQKD88)&P/OLQKD8)&P/OLQKD6
8)&P/OLQKD58)&P/OLQKD48)&P/OLQKD38)&POLQKD28)&P/OLQKD1 8)&
mL e linha 9, 1008)&P/OLQKDFRQWUROHQHJDWLYROHLWHVHPFRQWDPLQDomR3$,1(/%±$QiOLVHGRVIUDJPHQWRVGH
GLJHVWmRHQ]LPiWLFDSRUHOHWURIRUHVHHPJHOGHDJDURVHQDVFRQFHQWUDo}HVGH8 a 101 8)&P//LQKD3SDGUmRGH
WDPDQKRGHIUDJPHQWRSE3URPHJD/LQKDVD±KGLJHVWmRFRPDHQ]LPDBstEII (245pb, 125pb, 80pbHOLQKDVL±RFRP
DHQ]LPDHaeIII (150pb, 140pb, 70pb).
DISCUSSÃO
2 PpWRGR GH GLDJQyVWLFR PDLV FRQ¿iYHO
para doenças transmitidas por microorganismos
p VHP G~YLGD R LVRODPHQWR H LGHQWL¿FDomR GR
DJHQWHHWLROyJLFRDSDUWLUGHOHV}HVRULXQGDVGRV
DQLPDLV GRHQWHV 1R HQWDQWR HP WXEHUFXORVH
ERYLQD HPERUD D FXOWXUD EDFWHULROyJLFD DLQGD
UHSUHVHQWHRSDGUmRRXURGHGLDJQyVWLFRDGL¿FXOGDGHGHREWHQomRGHDPRVWUDVDHVFDVVH]GH
M. bovisQDVVHFUHo}HVERYLQDVHRWHPSRQHFHVViULRSDUDVHREWHURVUHVXOWDGRVTXHPXLWDVYH]HV
SRGHPUHVXOWDUHPIDOVRVQHJDWLYRV'8)),(/'
HWDOWRUQDPSRXFRYLiYHOVXDXWLOL]DomR
URWLQHLUD(PYLUWXGHGDLQH[LVWrQFLDGHH¿FLHQWHV
PpWRGRV GH GLDJQyVWLFR GLUHWR TXH SRVVDP VHU
XWLOL]DGRV QD DQiOLVH GH XP JUDQGH Q~PHUR GH
DPRVWUDVDOJXQVHVWXGRVYrPVHQGRIHLWRVFRPR
intuito de desenvolver métodos moleculares para
GHWHFWDUGHIRUPDGLUHWDDSUHVHQoDGRM. bovis
HPDPRVWUDVFOtQLFDVHDVVLPLGHQWL¿FDUGHIRU-
ma mais precisa, animais e produtos de origem
animal contaminados.
ROMERO et al. (1999), comparando a
WpFQLFDGH3&5FRPRVWHVWHVGHFXOWXUDHWXEHUFXOLQL]DomRFRQFOXtUDPTXHRPpWRGRPROHFXODU
pPDLVHVSHFt¿FRHVHQVtYHOSDUDGHWHFWDULQIHFo}HVDVVRFLDGDVFRPM. bovis em sangue, leite
HPXFRQDVDO$SHUIRUPDQFHGD3&5WDPEpP
MiIRLFRPSDUDGDFRPDQHFURSVLDHHQFRQWURX
VH XPD FRUUHODomR GH FRP RV WHVWHV HP
DPRVWUDVGHOLQIRQRGRV9,7$/(HWDO$
WpFQLFDGH3&5IRLWDPEpPXWLOL]DGDFRPVXFHVVR
na detecção de M. bovis em amostras de tecido
:$5'6HWDOPHVPRTXDQGRHPEHELGRVHPSDUD¿QD0,//(5HWDO
$LGHQWL¿FDomRGRFRPSOH[RM. tuberculosis HPDPRVWUDVFOtQLFDVGHERYLQRVWXEHUFXORVRV
é importante para os procedimentos veterinários,
pois pode indicar a presença de M. bovis ou alguPDRXWUDHVSpFLHGHVVHFRPSOH[RFDSD]GHFDXVDU
GRHQoDDRKRPHP(QWUHWDQWRDGLIHUHQFLDomR
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
1030
FIGUEIREDO, E. E. de S. et al.
entre as espécies do complexo M. tuberculosis
QmRVHID]QHFHVViULDQDPDLRUSDUWHGRVSURFHdimentos veterinários (COLLINS et al., 1994),
XPDYH]TXHRM. bovispRDJHQWHHWLROyJLFRGD
WXEHUFXORVHERYLQDHDLGHQWL¿FDomRGRFRPSOH[R
M. tuberculosisHPDPRVWUDVFOtQLFDVVHUHIHULUi
jSUHVHQoDGREDFLORERYLQR$LGHQWL¿FDomRGR
complexo em leite indica a presença de agente
LQIHFFLRVR FDSD] GH FDXVDU GRHQoD WXEHUFXORVH
extrapulmonar) ao homem.
Neste estudo, demonstrou-se experimenWDOPHQWH TXH D DQiOLVH GH OHLWH SRU 35$ p XPD
WpFQLFD VHQVtYHO HVSHFt¿FD GH UiSLGD H[HFXomR
HSHUPLWHGHWHFWDUHLGHQWL¿FDUDSUHVHQoDGRM.
bovis DWUDYpVGRSHU¿OFDUDFWHUtVWLFRGRVIUDJPHQWRV GH UHVWULomR GDV PLFREDFWpULDVGR FRPSOH[R
M. tuberculosis PHVPR TXDQGR SUHVHQWHV HP
FRQFHQWUDo}HVPXLWREDL[DV$OpPGLVVRFRPRGHmonstrado, não houve a necessidade de isolamento
prévio dos microrganismos a serem analisados. O
PpWRGRGHH[WUDomRGH'1$GHPLFREDFWpLUDVIRL
DSOLFDGRGLUHWDPHQWHQROHLWHWHQGRVLGRREWLGRV
PROGHVGH'1$FDSD]HVGHVHUHPDPSOL¿FDGRV
pela PCR.
7DLV UHVXOWDGRV PRVWUDP TXH p SRVVtYHO
LGHQWL¿FDUM. bovis diretamente a partir de uma
PDWUL]FRPSOH[DFRPRROHLWHWRUQDQGRRSURFHVVR
GHLGHQWL¿FDomRUiSLGRHH¿FLHQWH,VVRSRUTXHVH
WUDWDGHPpWRGRTXHQmRGHSHQGHGRSURFHGLPHQWR
GHGHVFRQWDPLQDomRGDDPRVWUDMiTXHHVWHHPDWp
GRVFDVRVQmRpEHPVXFHGLGR$OpPGLVVR
FRPDPHWRGRORJLDGHH[WUDomRGH'1$XWLOL]DGD
±EDUDWDHGHIiFLOH[HFXomR±IRLSRVVtYHOREWHU
XPDERDVHQVLELOLGDGHGRPpWRGRGH3&57DLV
FDUDFWHUtVWLFDVVmRLPSRUWDQWHVQDSRSXODUL]DomR
GD PHWRGRORJLD PROHFXODU SDUD LGHQWL¿FDomR GH
Mycobacterium.
CONCLUSÃO
$35$SDGURQL]DGDSDUDDQDOLVDUOHLWHSHUmitiu a detecção de M. bovis em uma concentração
PtQLPDGH8)&P/HPXPGLDGHWUDEDOKR
Diante dos resultados apresentados, trata-se de
WpFQLFD TXH SHUPLWH DX[LOLDU HRX WRUQDUVH XPD
DOWHUQDWLYDDRVWUDEDOKRVRVHGHPRUDGRVPpWRGRV
PLFURELROyJLFRV H ELRTXtPLFRV GH LGHQWL¿FDomR
Ciência Animal Brasileira, v. 9, n. 4, p. 1023-1033, out./dez. 2008
de M. bovis GXUDQWH HVWXGRV HSLGHPLROyJLFRV
SULQFLSDOPHQWH HP UHJL}HV FRQVLGHUDGDV GH DOWR
risco de transmissão.
(VWD WpFQLFD HPSUHJDGD H RWLPL]DGD HP
ODERUDWyULRSDUDLGHQWL¿FDUDSUHVHQoDM. bovis em
leite proveniente de vacas PPD +, representa uma
FRQWULEXLomRDRVHVIRUoRVIHLWRVSDUDDHUUDGLFDomR
GDWXEHUFXORVHERYLQDQRSDtV
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of Veterinary Science, YQS
$5$Ò-2&3/(,7(&4)35,1&(.$-25*(
.6* 26Ï5,2 $/$5 Mycobacterium bovis
LGHQWL¿FDWLRQ E\ D PROHFXODU PHWKRG IURP SRVWPRUWHP
LQVSHFWHGFDWWOHREWDLQHGLQDEDWWRLUVRI0DWR*URVVRGR6XO
%UD]LOMemórias do Instituto Oswaldo Cruz, v. 100, n.
S
$6+)25''$:+,71(<(5$*+81$7+$13
&26,9, 2 (SLGHPLRORJ\ RI VHOHFWHG P\FREDFWHULD WKDW
LQIHFW KXPDQV DQG RWKHU DQLPDOV 5HYXH 6FLHQWL¿TXH HW
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=$1,1, 0 6 6$/$6 & ( $ SQF$ SRO\PRUSKLVP
WR GLIIHUHQWLDWH EHWZHHQ Mycobacterium bovis and
Mycobacterium tuberculosis. Molecular and Cellular
ProbesYS
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$EDVWHFLPHQWR 3URJUDPD 1DFLRQDO GH &RQWUROH H
(UUDGLFDomRGH%UXFHORVHH7XEHUFXORVH±31&(%7
Disponível em: <ZZZDJULFXOWXUDJRYEr>. Acesso em: 4
mar. 2008.
CLARRIDGE, J.E6+$:$55M6+,11,&.7M
PLIKAYTIS, B.B /DUJHVFDOH XVH RI SRO\PHUDVH FKDLQ
UHDFWLRQIRUGHWHFWLRQRI0\FREDFWHULXPWXEHUFXORVLVLQD
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&2//,16 ' 0 5$')25' $ - /,6/( * :
%,//0$1 - + 'LDJQRVLV DQG HSLGHPLRORJ\ RI ERYLQH
WXEHUFXORVLV XVLQJ PROHFXODU ELRORJLFDO DSSURDFKHV
Veterinary Microbiology, v. 40, p. 83-94, 1994.
&2//,16'067(3+(16'0,GHQWL¿FDWLRQRIDQ
Detecção do complexo Mycobacterium tuberculosis no leite pela reação em cadeia da polimerase ...
LQVHUWLRQVHTXHQFH,6LQMycobacterium bovis. FEMS
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0LFURDUUD\ Journal of Clinical Microbiology, v. 41, p.
2605-2615, 2003.
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p. 53-63, 1994.
&255($ : 0 &255($ & 1 0 Enfermidades
infecciosas dos mamíferos domésticos6mR3DXOR9DUHOD
1981.
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Mycobacterium bovis LQIHFWLRQVeterinary Microbiology,
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Mycobacterium bovis in developing countries. Emerging
Infectious DiseasesYQS±
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Microbiology, v. 26, p. 1349-1352, 1988.
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of Microbiological Methods, v. 61, p. 193-199, 2005.
),*8(,5('2 ((6 *2/,1(//, / 6,/9$ -7
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GD WUDQVPLVVmR GD WXEHUFXORVH ERYLQD DR KRPHP DWUDYpV
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\HDUVRIH[SHULHQFHMemórias do Instituto Oswaldo Cruz,
v. 3, p. 801-805, 1998.
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Queensland. Australian Veterinary Journal, v. 66, p.
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