Dose response relations for gastro-instestinal pathogens in an animal model | RIVM
34
0
0
Hele tekst
(2) SDJHRI. 5,90UHSRUW. $EVWUDFW $OWKRXJKGRVH-UHVSRQVHPRGHOVSOD\DQHVVHQWLDOUROHLQTXDQWLWDWLYHPLFURELRORJLFDOULVN DVVHVVPHQWDSSURSULDWHDYDLODEOHGDWDIRUWKLVDVVHVVPHQWDUHVFDUFH6LQFHKXPDQYROXQWHHU VWXGLHVSURYLGHRQO\OLPLWHGLQIRUPDWLRQDQDQLPDOPRGHOLVEHLQJGHYHORSHGWRVWXG\ LQIHFWLRQZLWKKXPDQHQWHURSDWKRJHQLFEDFWHULD7KHPRGHOXVHGDGXOWPDOH:8UDWV H[SRVHGWRGLIIHUHQWGRVHVRIWKUHHHQWHURSDWKRJHQLFEDFWHULD 6DOPRQHOODHQWHULFDVHURYDU (QWHULWLGLV(VFKHULFKLDFROL2DQG&DPS\OREDFWHUMHMXQL
(3) DIWHURYHUQLJKWVWDUYDWLRQDQG QHXWUDOLVDWLRQRIJDVWULFDFLG$UHODWLRQVKLSEHWZHHQWKHGRVHDQGWKHSUREDELOLW\RILQIHFWLRQ ZDV GHPRQVWUDWHGIRUDOOWKUHHEDFWHULDZLWK& MHMXQLEHLQJPRUHLQIHFWLRXVWKDQ( FROL 2DQG6(QWHULWLGLVWKHOHDVWLQIHFWLRXVRIDOOWKUHH6 (QWHULWLGLVLQGXFHGDV\VWHPLF LQIHFWLRQDQGGRVHGHSHQGHQWHIIHFWVZHUHREVHUYHGIRUVRPHKDHPDWRORJLFDOSDUDPHWHUV QRWDEO\DQLQFUHDVHLQQHXWURSKLOV
(4) DVZHOODVIRUKLVWRSDWKRORJLFDOFKDQJHVLQWKHJDVWUR LQWHVWLQDOWUDFWDQGWKHGHOD\HGW\SHK\SHUVHQVLWLYLW\UHDFWLRQ7KHH[SHULPHQWVXVLQJ6 (QWHULWLGLVVKRZHGJRRGUHSURGXFLELOLW\ZKHUHDVWKHUHSURGXFLELOLW\IRUWKHH[SHULPHQWVZLWK &MHMXQLZDVSRRU.
(5) 5,90UHSRUW. SDJHRI. 3UHIDFH 7KHH[SHULPHQWVLQWKLVUHSRUWLQYROYHGWKHZRUNRIPDQ\SHUVRQVLQGLIIHUHQWODERUDWRULHV +DQV6WURRWPDQ0DULVNDYDQ'LMN'LUN(OEHUWVDQG%HUWYDQ0LGGHODDU &'/
(6) ZHUH UHVSRQVLEOH IRUDQLPDOH[SHULPHQWV&RHQ0RROHQEHHNSHUIRUPHGVHFWLRQRIWKHDQLPDOVDQG 3DXO5RKROO /3,
(7) ZDVUHVSRQVLEOHIRUKLVWRWHFKQLTXH+HQQ\/RHQGHUVORRWDQG6DQGUDGH :DDO SHUIRUPHGWKHKLVWRWHFKQLFDOZRUN<YRQQH:DOOEULQN /3,
(8) SDUWLFLSDWHGLQ KDHPDWRORJLFDODQDO\VHV(OOHQ'HOIJRXYDQ$VFKDQG:LOPD5LWPHHVWHU 0*%
(9) ZHUH LQYROYHGLQWKHPLFURELRORJLFDODQDO\VHV:LP-DQVHQDQG1DQYDQ/HHXZHQSURYLGHG EDFWHULDOVWUDLQV-RNHYDQGHU*LHVVHQ 0*%
(10) 0DULRQ.RRSPDQV /,2
(11) -RRS6FKHOOHNHQV DQG 5RQ%RRW /,6
(12) SURYLGHGKHOSIXODGYLFHRQELRPHGLFDODVSHFWVDQG:RXW6OREDQG3HWHU 7HXQLVRQVWDWLVWLFDODVSHFWVRIWKHZRUN.
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
(14) 5HVXOWV ,QRFXOXPFXOWXUHV &OLQLFDOREVHUYDWLRQV 0LFURELRORJ\ 'HOD\HGW\SHK\SHUVHQVLWLYLW\UHDFWLRQ '7+
(15) +DHPDWRORJ\ 3DWKRORJ\ &RQFOXVLRQ 5HIHUHQFHV $SSHQGL[0DLOLQJOLVW $SSHQGL[,QGLYLGXDOKLVWRSDWKRORJLFDOGDWD6(QWHULWLGLVH[SHULPHQW$$3 $SSHQGL[,QGLYLGXDOKLVWRSDWKRORJLFDOGDWD6(QWHULWLGLVH[SHULPHQW$$3 .
(16) 5,90UHSRUW. SDJHRI. 6DPHQYDWWLQJ. 'RVLVUHVSRQVPRGHOOHQ]LMQHHQEHODQJULMNRQGHUGHHOYDQGHNZDQWLWDWLHYHPLFURELRORJLVFKH ULVLFRDQDO\VHPDDUHU]LMQVOHFKWVZHLQLJEUXLNEDUHNZDQWLWDWLHYHJHJHYHQV([SHULPHQWHQ PHW YULMZLOOLJHUVJHYHQVOHFKWVEHSHUNWHLQIRUPDWLH'DDURPZRUGWHHQGLHUPRGHOYRRU LQIHFWLHPHWHQWHURSDWKRJHQHEDFWHULsQRQWZLNNHOG,QHHQYRRUJDDQGUDSSRUW *DUVVHQHWDO
(17) ZHUGEHVFKUHYHQGDWGULHHQWHURSDWKRJHQHEDFWHULsQ 6DOPRQHOODHQWHULFDVHURYDU (QWHULWLGLV(VFKHULFKLDFROL2HQ&DPS\OREDFWHUMHMXQL
(18) LQVWDDWZDUHQKHWGDUPNDQDDO YDQYROZDVVHQPDQQHOLMNH:8UDWWHQWHNRORQLVHUHQQDEHVPHWWLQJYDQGHGLHUHQPHWKRJH DDQWDOOHQEDFWHULsQ FIXGLHU
(19) YLDHHQPDDJVRQGH'HNRORQLVDWLHZHUGEHYRUGHUG GRRU RYHUQDFKWYDVWHQHQQHXWUDOLVDWLHYDQPDDJ]XXUPHWQDWULXPELFDUERQDDW ,QGLWUDSSRUWZRUGWRQGHU]RHNEHVFKUHYHQQDDUGRVLVUHVSRQVUHODWLHVLQGLWUDWWHQPRGHO 'DDUWRHZHUGHQGHGLHUHQEHVPHWPHWYHUVFKLOOHQGHDDQWDOOHQEDFWHULsQ FIXGLHU
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
(21) PHWGHYHUWUDDJGW\SHRYHUJHYRHOLJKHLGVUHDFWLH 279
(22) HQPHW KLVWRSDWKRORJLVFKHYHUDQGHULQJHQLQKHWGDUPNDQDDO%LMEORRWVWHOOLQJYDQGHGLHUHQDDQODJH DDQWDOOHQ6 (QWHULWLGLVZHUGJHHQIHFDOHXLWVFKHLGLQJJHYRQGHQPDDUZHOV\VWHPLVFKH LQIHFWLH]RDOVEOHHNXLWLVRODWLHYDQGHEDFWHULsQXLWGHPHVHQWHULDOHO\PINOLHUHQHQXLWGH PLOW'HSRVLWLHYH279UHDFWLHGXLGWRSLQGXFWLHYDQ V\VWHPLVFKH
(23) FHOOXODLUHLPPXQLWHLW WHJHQGHEDFWHULH . . . .
(24) SDJHRI. 5,90UHSRUW. 6XPPDU\. 'RVHUHVSRQVHPRGHOVDUHDQHVVHQWLDOSDUWRITXDQWLWDWLYHPLFURELRORJLFDOULVNDVVHVVPHQW EXWQRWPDQ\DSSURSULDWHTXDQWLWDWLYHGDWDDUHDYDLODEOHIRUWKLVSXUSRVH+XPDQYROXQWHHU VWXGLHVSURYLGHRQO\OLPLWHGLQIRUPDWLRQ7KHUHIRUHDQDQLPDOPRGHOIRULQIHFWLRQZLWK KXPDQHQWHURSDWKRJHQLFEDFWHULDLVEHLQJGHYHORSHG$SUHYLRXVUHSRUW *DUVVHQHWDO
(25) GHVFULEHVWKDWWKUHHHQWHURSDWKRJHQLFEDFWHULD 6DOPRQHOODHQWHULFDVHURYDU(QWHULWLGLV (VFKHULFKLDFROL2DQG&DPS\OREDFWHUMHMXQL
(26) ZHUHDEOHWRFRORQLVHWKHLQWHVWLQDOWUDFWRI DGXOWPDOH:8UDWVDIWHULQRFXODWLRQE\JDYDJHRIKLJKGRVHV FIXDQLPDO
(27) 2YHUQLJKWVWDUYDWLRQDQGQHXWUDOLVDWLRQRIJDVWULFDFLGZLWKVRGLXPELFDUERQDWHSURPRWHG FRORQLVDWLRQ 7KLVUHSRUWGHVFULEHVLQYHVWLJDWLRQVLQGRVHUHVSRQVHUHODWLRQVXVLQJWKHUDWPRGHO$QLPDOV ZHUH LQRFXODWHGZLWKGLIIHUHQWGRVHVRIEDFWHULD FIXDQLPDO
(28) XQGHUVLPLODU FRQGLWLRQV7KHWKUHHHQWHURSDWKRJHQLFVSHFLHVZHUHDEOHWRFRORQLVHWKHUDWV¶LQWHVWLQDOWUDFW LQ FRQWUDVWWRWKHQHJDWLYHFRQWUROVWUDLQ(FROL:*:KHQKLJKGRVHVZHUHDGPLQLVWHUHG6 (QWHULWLGLVLQGXFHGVHYHUHV\VWHPLFHIIHFWVEXW& MHMXQLRU ( FROL2FRORQLVHGWKH LQWHVWLQDOWUDFWZLWKRXWVLJQVRIFOLQLFDOLOOQHVV$UHODWLRQVKLSEHWZHHQWKHGRVHDQGWKH SUREDELOLW\RILQIHFWLRQZDVGHPRQVWUDWHGIRUDOOWKUHHEDFWHULDZLWK& MHMXQLEHLQJPRUH LQIHFWLRXVWKDQ( FROL2DQG6 (QWHULWLGLVEHLQJOHDVWLQIHFWLRXV)RU6 (QWHULWLGLVD GRVHUHVSRQVHUHODWLRQZDVDOVRGHPRQVWUDWHGIRUVRPHKDHPDWRORJLFDOSDUDPHWHUV QRWDEO\ DQ LQFUHDVHLQQHXWURSKLOV
(29) IRUGHOD\HGW\SHK\SHUVHQVLWLYLW\ '7+
(30) UHDFWLRQDQGIRU KLVWRSDWKRORJLFDOFKDQJHVLQWKHLQWHVWLQDOWUDFW$QLPDOVFKDOOHQJHGZLWKORZGRVHV FIXDQLPDO
(31) RI6 (QWHULWLGLVGLGQRWVKHGWKHEDFWHULDLQWKHLUIDHFHVEXWV\VWHPLFLQIHFWLRQ ZDV GHPRQVWUDWHGE\LVRODWLRQRIWKHEDFWHULDIURPPHVHQWHULFO\PSKQRGHVDQGVSOHHQ7KH SRVLWLYH'7+UHDFWLRQLQGLFDWHGLQGXFWLRQRI V\VWHPLF
(32) FHOOXODULPPXQLW\DJDLQVW6 (QWHULWLGLV . . . .
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
(34) DQGWKHSUREDELOLW\RIDQHIIHFWVXFKDV LQIHFWLRQRUGLVHDVH 'RVHUHVSRQVHPRGHOVFDQEHEDVHGRQREVHUYDWLRQDORUH[SHULPHQWDOGDWD2EVHUYDWLRQDO GDWD XVXDOO\IURPIRRGRUZDWHUERUQHRXWEUHDNV
(35) KDYHWKHDGYDQWDJHWKDWWKH\DUHEDVHGRQ DFWXDOVLWXDWLRQVEXWDUHH[WUHPHO\OLPLWHGLQDYDLODELOLW\IRUVHYHUDOUHDVRQV7KHGRVHPD\ EHWRRORZWREHPHDVXUHGDFFXUDWHO\VXFKDVLQGULQNLQJZDWHUWKHFRQWDPLQDWLRQPD\EH UDUH VXFKDVZLWKIRRGVWXIIVRUVDPSOHVRIWKHFDXVDOSURGXFWPD\VLPSO\QRWEHDYDLODEOH )XUWKHUPRUHWKHVL]HRIWKHH[SRVHGSRSXODWLRQLVRIWHQQRWNQRZQ([SHULPHQWDOGDWDKDYH WKHDGYDQWDJHWKDWWKH\DUHREWDLQHGXQGHUZHOOFRQWUROOHGFRQGLWLRQVDQGFDQWKHUHIRUHEH VXEMHFWHGWRGHWDLOHGPDWKHPDWLFDODQDO\VLV 7HXQLVHWDO
(36) 7\SLFDOO\KHDOWK\ YROXQWHHUVDUHH[SRVHGWRGLIIHUHQWGRVHVRIHQWHULFSDWKRJHQVWKDWKDYHEHHQPDLQWDLQHGDQG FXOWXUHGLQODERUDWRU\FRQGLWLRQV7KHYROXQWHHUVDUHWKHQFORVHO\PRQLWRUHGIRUVLJQVRI LQIHFWLRQ H[FUHWLRQVHURFRQYHUVLRQ
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
(38) RQWKHVXVFHSWLELOLW\RIWKHKRVWDQGIDFWRUVVXFKDVEDFWHULDOVWUHVV SURWHLQVSURWHFWLRQE\IDWW\VXEVWUDWHVHWFRQWKHLQIHFWLYLW\RIWKHSDWKRJHQ$VDQH[WVWHS ZH SODQWRGHYHORSNLQHWLFPRGHOVRIWKHLQIHFWLRQSURFHVVWKDWGHVFULEHWKHG\QDPLFVRIWKH KRVWSDWKRJHQLQWHUDFWLRQLQWKHDOLPHQWDU\WUDFWIRUZKLFKWKHDQLPDOPRGHOVVKRXOGSURYLGH LQVLJKWLQLPSRUWDQWPHFKDQLVPVDQGSDUDPHWHUHVWLPDWHV ,QDILUVWVHULHVRIH[SHULPHQWV *DUVVHQHWDO
(39) LWZDVFRQFOXGHGWKDWDIWHU QHXWUDOLVDWLRQRIJDVWULFDFLGZLWKVRGLXPELFDUERQDWH\RXQJDGXOWPDOH:8UDWVZHUH VXVFHSWLEOH WRRUDOLQIHFWLRQZLWKKLJKGRVHV FIXSHUDQLPDO
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
(41) SDJHRI. 5,90UHSRUW. 6XVFHSWLELOLW\RIUDWVWRGLIIHUHQWGRVHVRI HQWHURSDWKRJHQLFEDFWHULDE\RUDOH[SRVXUH ,QWURGXFWLRQ. 7RHYDOXDWHGRVHGHSHQGHQWHIIHFWVRIH[SRVXUHRIDGXOWUDWVWRWKUHHHQWHURSDWKRJHQLF EDFWHULD 6DOPRQHOODHQWHULFDVHURYDU(QWHULWLGLV(VFKHULFKLDFROL2DQG&DPS\OREDFWHU MHMXQL
(42) WKHDQLPDOVZHUHLQRFXODWHGZLWKGLIIHUHQWGRVHVEHWZHHQ DQG FIXSHUDQLPDO XQGHU WKHVDPHH[SHULPHQWDOFRQGLWLRQVDVSUHYLRXVO\UHSRUWHG *DUVVHQHWDO
(43) . . 0DWHULDOVDQGPHWKRGV $QLPDOV. 6SHFLILFSDWKRJHQIUHH 63)
(44) PDOH:LVWDU8QLOHYHU :8
(45) UDWVZHUHREWDLQHGIURPWKH EUHHGLQJFRORQ\DWWKH1DWLRQDO,QVWLWXWHRI3XEOLF+HDOWKDQGWKH(QYLURQPHQW%LOWKRYHQ 7KH1HWKHUODQGV7KHDQLPDOV ZHHNVRIDJHZHUHKRXVHGLQGLYLGXDOO\LQPDFURORQ FDJHVRQHZHHNSULRUWRWKHVWDUWRIWKHH[SHULPHQWV'ULQNLQJZDWHUDQGFRQYHQWLRQDOGLHW 50+%+RSH)DUPV%9:RHUGHQ7KH1HWKHUODQGV
(46) ZHUHSURYLGHGDGOLELWXP 7KH EUHHGLQJFRORQ\RIWKHDQLPDOVZDVSUHVFUHHQHGPRQLWRUHGIRUHQGRJHQRXVSDWKRJHQLF YLUXVHVDQGEDFWHULDDQGZDVIRXQGQHJDWLYH. %DFWHULDOVWUDLQV. (VFKHULFKLDFROL VWUDLQQXPEHUVHURW\SH2.+DQLVRODWHIURPDSDWLHQWZLWK +DHPRO\WLF8UHPLF6\QGURPHZLWKHDHDQGVW[JHQHV 6DOPRQHOODHQWHULFDVHURYDU(QWHULWLGLVVWUDLQQXPEHU SDWLHQWLVRODWH RULJLQ5,90
(47) &DPS\OREDFWHUMHMXQL VWUDLQQXPEHU%VHURW\SH2 IURPFKLFNHQIDHFHV0HGHPDHW DO
(48) DQG ( FROL VWUDLQQXPEHU:* DQDOLGL[LFDFLGUHVLVWDQWGHULYDWLYHRI( FROL& +DYHODDUDQG +RJHERRP
(49) ZDVXVHGDVDQHJDWLYHFRQWURO )URPDOOVWUDLQVD&FROOHFWLRQZDVPDGHE\SXUHFXOWXULQJRQ%UDLQ+HDUW,QIXVLRQ %+,
(50) DJDU KDW&
(51) DQGLQRFXODWLQJDVLQJOHFRORQ\LQ%+,LQFXEDWHGIRUK DW & &MHMXQLZDVLQFXEDWHGXQGHUPLFURDHURSKLOLFFRQGLWLRQVDW&IRUK
(52) $IWHU LQFXEDWLRQPORIWKHFXOWXUHZDVDGGHGWRFU\RWXEHVILOOHGZLWKJODVVEHDGVDQGPORI JO\FHURO'LUHFWO\DIWHUDGGLQJWKHFXOWXUHVWKHFU\RWXEHVZHUHSODFHGLQD&IUHH]HU. ,QRFXOXPFXOWXUHV. %RWK( FROLVWUDLQVDQG6 (QWHULWLGLVZHUHLQRFXODWHGIURPWKH&FROOHFWLRQLQ%+,DQG LQFXEDWHGDW&IRUK$IWHULQFXEDWLRQPORIHDFKFXOWXUHZDVFHQWULIXJHGDW [ J IRU PLQDW&7KHVXSHUQDWDQWZDVGLVFDUGHGDQGWKHSHOOHWZDVUHVXVSHQGHGLQ PO SK\VLRORJLFDOVDOLQH 36
(53) IROORZHGE\UHFHQWULIXJDWLRQ$JDLQWKHVXSHUQDWDQWZDV GLVFDUGHGDQGWKHSHOOHWZDVUHVXVSHQGHGLQDYROXPHRIPO367KHFHOOVXVSHQVLRQDQG VHULDO
(54) GLOXWLRQVLQ36ZHUHGHOLYHUHGDWWKHDQLPDOGHSDUWPHQWRQPHOWLQJLFH& MHMXQLZDV WUHDWHGVLPLODUO\EXWLQFXEDWHGXQGHUPLFURDHURSKLOLFFRQGLWLRQVDW&ZKLOH VKDNLQJDWUSPIRUK)RUZDVKLQJDQGFRQFHQWUDWLQJWKHFXOWXUHZDVILOWHUHGWKURXJK DQ PPHPEUDQHILOWHU 1DOJHQHPO
(55) LQVWHDGRIFHQWULIXJDWLRQ. . 7KLVH[SHULPHQWLVUHJLVWHUHGDV$$3 .
(56) 5,90UHSRUW. SDJH RI. 'LUHFWO\EHIRUHDGPLQLVWUDWLRQWRWKHDQLPDOVPORIHDFKEDFWHULDOVXVSHQVLRQZDVPL[HG ZLWKPORIDVROXWLRQRI ZY
(57) 1D+&2 $IWHUDGPLQLVWUDWLRQ
(58) WKHLQRFXOXP FXOWXUHVZHUHWUDQVSRUWHGWRWKHPLFURELRORJLFDOODERUDWRU\RQPHOWLQJLFHIRUSODWHFRXQWVRQ VKHHSEORRGDJDU LQFXEDWHGDVDERYH
(59) . ([SHULPHQWDOGHVLJQ. (DFKDQLPDOZDVLPSODQWHGZLWKDWHPSHUDWXUHWUDQVSRQGHU %LR0HGLF'DWD6\VWHPV 6HDIRUG'HODZDUH8.
(60) VXEFXWDQHRXVO\LQWKHQHFN7KHWUDQVSRQGHUZDVFRGHGIRUWKH XQLTXH
(61) DQLPDOQXPEHUDQGZDVXVHGIRUERG\WHPSHUDWXUHGHWHFWLRQHDFKGD\DURXQG DP%RG\WHPSHUDWXUHDQGDQLPDOQXPEHUVZHUHUHJLVWHUHGXVLQJD%LRPHG3RFNHW 6FDQQHUIURP3OH[[(OVW7KH1HWKHUODQGV $IWHURQHZHHNRIUHVW LHDFFOLPDWLVDWLRQ
(62) WKHDQLPDOVZHUHVWDUYHGRYHUQLJKW ZDWHUDG OLELWXP
(63) $IWHUKRXUVRIVWDUYDWLRQPORIWKHEDFWHULDOVXVSHQVLRQVZDVRUDOO\ DGPLQLVWHUHGE\JDYDJH DQLPDOVSHUJURXSJURXSVSHUSDWKRJHQ
(64) 'LUHFWO\DIWHUJDYDJH GD\
(65) IRRGDQGZDWHUZDVSURYLGHGDGOLELWXP %ORRGVDPSOHVZHUHWDNHQYLDRUELWDSOH[XVSXQFWXUHXVLQJDFDSLOODU\XQGHUOLJKWHWKHU DQDHVWKHVLD RQGD\V±DQG 6 (QWHULWLGLVDQG(FROL2H[SHULPHQWV
(66) ,QWKH&MHMXQL VWXG\EORRGVDPSOHVZHUHREWDLQHGRQGD\V±DQG$SSUR[LPDWHO\PORIEORRGZDV REWDLQHGSHUVDPSOLQJWLPHSRLQW 'DLO\FOLQLFDOREVHUYDWLRQVZHUHPDGHZLWKUHIHUHQFHWRWKHVWDWXVRIJHQHUDOKHDOWKRIWKH DQLPDOV 6SHFLDODWWHQWLRQZDVSDLGWRWKHQDWXUHRIWKHIDHFHV7KHDQLPDOVZHUHZHLJKHGHDFK GD\ HDUO\LQWKHPRUQLQJ
(67) VWDUWLQJRQHGD\SULRUWRWKHRUDOLQRFXODWLRQ(DFKPRUQLQJIDHFHV ZDV REWDLQHGGLUHFWO\IURPHDFKUDWLQHDFKJURXS7KHIDHFHVZDVPDFURVFRSLFDOO\HYDOXDWHG DQG WHVWHGIRUPLFURELRORJ\WKHVDPHGD\ LQZHHNHQGV±GD\VDQG±VDPSOHVZHUHVWRUHGDW &DQGH[DPLQHGWKHQH[W0RQGD\±GD\
(68) 7KHDQLPDOVZHUHVDFULILFHGRQGD\ GD\IRU& MHMXQL
(69) DIWHURUDOLQRFXODWLRQE\EOHHGLQJ IURP WKHDEGRPLQDOODUJHYHVVHOVXQGHUQHPEXWDODQDHVWKHVLD LS
(70) 7KHFDHFXPRIHDFKUDWZDV ZHLJKHGDIWHUHXWKDQDVLDDQGFDHFXPZHLJKWVUHODWLYHWRERG\ZHLJKWZHUHFDOFXODWHG8ULQH ZDV REWDLQHGIURPWKHEODGGHUDQGVWRUHGDW&7KHHQWLUHH[SHULPHQWZDVGLYLGHGLQWR WKUHHFRKRUWVHDFKFRKRUWZLWKLWVRZQQHJDWLYHFRQWURO :*
(71) . )DHFDOPRLVWXUHFRQWHQW. $VDQLQGLFDWRUIRUGLDUUKRHDOOLNHHIIHFWVWKHPRLVWXUHFRQWHQWRIWKHIDHFHVZDVGHWHUPLQHG HDFK GD\E\FDOFXODWLRQIURPWKHZHLJKWORVVDIWHUGU\LQJ)RUWKLVSXUSRVHWKHVDPSOHVZHUH ZHLJKHGLQDJODVVFRQWDLQHUZLWKDLUWLJKWOLGDQGGULHGLQWKHRSHQFRQWDLQHUIRUKRXUDW &$IWHUFRROLQJLQDQH[VLFFDWRUZLWKVLOLFDJHOWKHFRQWDLQHUZDVFORVHGDQGZHLJKHG 7KLVSURFHGXUHZDVUHSHDWHGVHYHUDOWLPHVXQWLOFRQVWDQWZHLJKW. +DHPDWRORJ\. $VDQLQGLFDWRUIRU V\VWHPLF
(72) LQIHFWLRQKDHPDWRORJ\IRUHDFKUDWZDVSHUIRUPHGLQEORRG VDPSOHVDQWLFRDJXODWHGZLWK. ('7$REWDLQHGDWGD\DQGRU7KHKDHPDWRORJLFDO DQDO\VHVZHUHSHUIRUPHGXVLQJWKH+(DPXOWLVSHFLHVKDHPDWRORJ\DQDO\VHU %D\HU%9 0LMGUHFKW7KH1HWKHUODQGV
(73) ZLWKPXOWLVSHFLHVVRIWZDUHYHUVLRQ .
(74) SDJHRI. 5,90UHSRUW. 7KHIROORZLQJSDUDPHWHUVZHUHGHWHUPLQHG +DHPRJORELQFRQFHQWUDWLRQ +JE
(75) +DHPDWRFULW +FW
(76) 5HG%ORRGFHOO&RQFHQWUDWLRQ 5%&
(77) 0HDQ&HOO9ROXPH 0&9
(78) 0HDQ&HOO+DHPRJORELQ 0&+
(79) 0HDQ&HOO+DHPRJORELQ&RQF 0&+&
(80) 5HGEORRGFHOO'LVWULEXWLRQ:LGWK 5':
(81) +DHPRJORELQ'LVWULEXWLRQ:LGWK +':
(82) 3ODWHOHWV 3OW
(83) 0HDQSODWHOHWYROXPH 039
(84) :KLWH%ORRGFHOO&RQFHQWUDWLRQ :%&
(85) 'LIIHUHQWLDWLRQRIZKLWHEORRGFHOOV DQGDEVROXWHQXPEHUV
(86). 0LFURELRORJ\. )DHFDOVDPSOHVZHUHKRPRJHQLVHGDQGGLOXWHG ZY
(87) LQSHSWRQHSK\VLRORJLFDOVDOLQH 336
(88) DQGDSSURSULDWHGLOXWLRQVZHUHVSUHDGRQ%ULOOLDQW*UHHQ$JDU %*$
(89) IRU6 HQWHULWLGLV RQ6RUELWRO0F&RQNH\FHIL[LPHWHOOXULWHDJDU 6PDF&7
(90) IRU( FROL2RQ7U\SWRQH<HDVW ([WUDFW*OXFRVHDJDUZLWKQDOLGL[DFLGJPO 7<*QDO
(91) IRU( FROL:*DQGRQ%ROWRQ PHGLXPIRU& MHMXQL%7KH%*$6PDF&7DQG7<*QDOZHUHLQFXEDWHGDW&IRU KKDQGKUHVSHFWLYHO\7KH%ROWRQPHGLXPZDVLQFXEDWHGXQGHUPLFUR DHURSKLOLFFRQGLWLRQVIRUKDW& 0HVHQWHULFO\PSKQRGHVZHUHKRPRJHQLVHGLQ336E\XVLQJDQ8OWUD7KXUUD[$SSURSULDWH GLOXWLRQVZHUHVSUHDGSODWHGRQ%*$60$&&77<*QDODQG%ROWRQPHGLXPDQGLQFXEDWHG DV GHVFULEHGDERYH%ORRGVDPSOHV GD\
(92) ZHUHDQDO\VHGE\VSUHDGSODWLQJPOYROXPHV RQWKHVDPHPHGLD. 3DWKRORJ\. ,PPHGLDWHO\DIWHUH[VDQJXLQDWLRQWKHDEGRPHQDQGWKRUD[ZHUHLQVSHFWHGDQGWKHJDVWUR LQWHVWLQDOWUDFWZDVUHPRYHGDQGSURFHVVHG0DFURVFRSLFDEQRUPDOLWLHVZHUHUHFRUGHGDVZHOO DV JURVVGDWDSHULQWHVWLQDOVHJPHQW YROXPHDQGRXWHUFRORXUJXWDVVRFLDWHGO\PSKRLGWLVVXH TXDQWLW\DQGTXDOLW\RIFRQWHQWWKLFNQHVVRIZDOODVSHFWRIPXFRVD
(93) 7KHVWRPDFK VHJPHQWVRIWKHVPDOOLQWHVWLQH GXRGHQXPMHMXQXPDQGLOHXPHDFKZLWK3H\HUV¶SDWFK
(94) FDHFXPSUR[LPDODQGGLVWDOFRORQPHVHQWHULFO\PSKQRGHVOLYHUVSOHHQDQGWLVVXHRI PDFURVFRSLFDOO\DEQRUPDORUJDQVZHUHVDPSOHGDQGIL[HGLQSKRVSKDWHEXIIHUHG IRUPDOGHK\GH7KHJXWVHJPHQWVZHUHSURFHVVHGXVLQJWKH6ZLVVUROOWHFKQLTXH 0RROHQEHHN DQG 5XLWHQEHUJ
(95) $IWHUIL[DWLRQWKHWLVVXHVZHUHHPEHGGHGLQSDUDSODVWDQGP WKLFNVHFWLRQVZHUHSUHSDUHGDQGURXWLQHO\VWDLQHGZLWKKDHPDWR[\OLQDQGHRVLQ +(DOOGRVH JURXSV
(96) DQG3$6 FRQWURODQGKLJKHVWGRVHJURXS
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±WRGD\.
(98) 5,90UHSRUW. SDJHRI. 6 (QWHULWLGLV
(99) RUWRGD\ & MHMXQL
(100) 7KHGDWDIRUIDHFDOPRLVWXUHFRQWHQWZHUHXVHGIURP GD\±WRGD\ 6(QWHULWLGLV
(101) WRGD\ ( FROL2
(102) RUWRGD\ & MHMXQL
(103) :HOVK¶V DSSUR[LPDWHWHVWZDVDSSOLHGWRFRPSDUHWKHUHODWLYHFDHFXPZHLJKWRIHDFKRIWKHJURXSV ZLWKWKHFRQWUROJURXS:HDQDO\VHGWKHKDHPDWRORJLFDOGDWDIRUHDFKRIWKHEDFWHULDO LQIHFWLRQVE\PHDQVRIOLQHDUUHJUHVVLRQXVLQJWKHIROORZLQJPRGHO /RJ 5(63216(
(104) a b '$<g '$</RJ '26(
(105) 7KHYDULDEOH'$<PHDQVHLWKHUSULRUWRLQIHFWLRQ '$<
(106) RUSRVWLQIHFWLRQ '$<
(107) )RUWKH/RJ '26(
(108) WHUPZHXVHGDYDOXHRIIRUWKHFRQWUROJURXS,QWKLVPRGHOD VLJQLILFDQWGD\HIIHFWGXHWRH[SHULPHQWDOFRQGLWLRQVLVREVHUYHGLI DQGDGRVHUHODWHG HIIHFWLI . . .
(109) SDJHRI. 5,90UHSRUW. 5HVXOWV. ,QRFXOXPFXOWXUHV. 7DEOHVKRZVWKHSODWHFRXQWVRIGLIIHUHQWGLOXWLRQVRIWKHLQRFXOXPFXOWXUHRQDORJ VFDOH 7KHLQWHQGHG GLOXWLRQUDWLRZDVREWDLQHGZLWKWKHH[FHSWLRQRIWKHYHU\ORZGRVHVIRU 6 (QWHULWLGLVDQG& MHMXQL . 7DEOH0LFURELRORJLFDODQDO\VLV FRORQ\FRXQWSHUPOORJ LQEUDFNHWV
(110) RILQRFXOXPFXOWXUHV. 'RVHJURXS +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH 6HFRQGORZHVW /RZHVW &RQWURO :*
(111). 6 (QWHULWLGLV [
(112) [
(113) [
(114) [
(115) [
(116) [
(117) . (FROL2 [
(118) [
(119) [
(120) [
(121) [
(122) [
(123) . & MHMXQL [
(124) [
(125) [
(126) [
(127) [
(128) [
(129) . &OLQLFDOREVHUYDWLRQV. 7ZRRXWRIWKUHH6DOPRQHOODLQIHFWHGDQLPDOV KLJKHVWGRVH
(130) VKRZHGVHYHUHVLJQVRILOOQHVV ORZPXVFOHWHQVLRQFROGLQFUHDVHGQDVDOGLVFKDUJHUHGFUXVWVDURXQGH\HVDQGQRVHWLSV UXIIOHG IXU
(131) DWGD\VILYHDQGVL[DIWHULQIHFWLRQ,QPRVWJURXSVWKHERG\ZHLJKWLQFUHDVHG DIWHU LQRFXODWLRQ VHH)LJXUH
(132) ,QWKH6 (QWHULWLGLVKLJKHVWGRVHJURXSWKHUDWVORVWZHLJKW *UDP . 6 (QWHULWLGLV. &WUO . . *UDP . . *UDP . ( FROL. . . . . 'D\. &WUO . . 'D\. & MHMXQL. . . . . . . 'D\. &WUO . )LJXUH%RG\ZHLJKWVRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWRWKUHHHQWHURSDWKRJHQLF EDFWHULD(DFKV\PEROLQGLFDWHVWKHPHDQRILQGLYLGXDODQLPDOV/HJHQG ORJGRVHSHUDQLPDO &WUO FRQWURO :*
(133) . %RG\WHPSHUDWXUHVDQGIDHFDOPRLVWXUHFRQWHQWGLGQRWVKRZFRQVLVWHQWWUHQGVRYHUWLPHLQ UHODWLRQ ZLWKLQIHFWLRQ.
(134) 5,90UHSRUW. SDJHRI. 7KHFDHFXPZHLJKWVLQDOODQLPDOVZHUHOHVVWKDQRIWKHWRWDOERG\ZHLJKWLQGLFDWLQJQR PDMRUDEQRUPDOLW\LQWKHUDW¶VLQWHVWLQDOPLFURIORUD&RPSDUHGWRWKHFRQWUROJURXSVWKHUH ZDV DVLJQLILFDQWGLIIHUHQFHLQFDHFXPWRERG\ZHLJKWUDWLRLQVRPHJURXSV 6 (QWHULWLGLV KLJKHVWGRVHJURXS( FROL2VHFRQGKLJKHVWDQGVHFRQGORZHVWGRVHJURXSVVHH)LJXUH
(135) 3HUFHQW 3HUFHQW . ( FROL. . . . . 6 (QWHULWLGLV. . . . . . 3HUFHQW 'RVH . 'RVH & MHMXQL. . . . . . . . 'RVH. )LJXUH&DHFXPWRERG\ZHLJKWUDWLRRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWRWKUHH HQWHURSDWKRJHQLFEDFWHULD(DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDO DQLPDOV'RVH ORJGRVHSHUDQLPDO FRQWURO :*
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
(137) SDJHRI. 5,90UHSRUW /RJ FIX . 6 (QWHULWLGLV &WUO . /RJ FIX . ( FROL. . . &WUO . . 'D\. . /RJ . FIX. 'D\ & MHMXQL &WUO . . . . . 'D\. )LJXUH&RORQ\FRXQWVLQWKHIDHFHVRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWRWKUHH HQWHURSDWKRJHQLFEDFWHULD(DFKV\PEROLQGLFDWHVWKHPHDQRILQGLYLGXDODQLPDOV/HJHQG ORJ GRVHSHUDQLPDO&WUO FRQWURO :*
(138) . )RUSXUSRVHVRIGRVHUHVSRQVHPRGHOOLQJDQGFRPSDULVRQZLWKKXPDQYROXQWHHUH[SHULPHQWV LWLVDOVRRILQWHUHVWWRLQWHUSUHWWKHGDWDEDVHGRQWZRRXWFRPHV WKHDQLPDOZDVLQIHFWHGRU QRWLQIHFWHG
(139) )RUWKLVSXUSRVHLQIHFWLRQFDQEHGHILQHGDVHTXLYDOHQWWRIDHFDOVKHGGLQJDW DQ\PRPHQWLQWLPH QRWHWKDWLQWKHFRQWUROJURXSVQREDFWHULDZHUHGHWHFWHGLQIDHFHVDWDQ\ PRPHQW
(140) ,QORZHUGRVHJURXSVKRZHYHUFRXQWVRIFIXSHUJUDP LHRQHVSHFLILFFRORQ\ RQRQHRIWZRGXSOLFDWHSODWHV
(141) ZHUHVRPHWLPHVVHHQDQGWKHVHFRXOGEHDWWULEXWHGWRWUDQVLHQW FRORQLVDWLRQRQO\7KHUHIRUHZHKDYHDGRSWHGDVRPHZKDWKLJKHUWKUHVKROGWRGHILQH LQIHFWLRQ IDHFDOH[FUHWLRQRIFIXSHUJUDPRQDWOHDVWWZRGD\VDVWKHFULWHULRQIRU LQIHFWLRQ LHDFWLYHPXOWLSOLFDWLRQRIWKHSDWKRJHQLQWKHJDVWURLQWHVWLQDOWUDFW
(142) 7KHUHVXOWV FDQ WKHQEHVXPPDULVHGDVLQ7DEOH,WVKRZVWKDW& MHMXQLZDV WKHPRVWLQIHFWLRXVRIWKH WKUHHSDWKRJHQVWHVWHGIROORZHGE\(FROL2ZLWK6 (QWHULWLGLVEHLQJOHDVWLQIHFWLRXV 7DEOH,QIHFWLRQ GHILQHGE\IDHFDOH[FUHWLRQ
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
(144) 5,90UHSRUW. SDJHRI. 7DEOH3DWKRJHQLFEDFWHULD FIXSHUJUDP
(145) LQPHVHQWHULFO\PSKQRGHVRIDGXOWPDOH:8UDWVDIWHU RUDOH[SRVXUHWRGLIIHUHQWGRVHV. 'RVHJURXS. +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH 6HFRQGORZHVW /RZHVW. 6 (QWHULWLGLV GD\
(146) [
(147) [
(148) [
(149)
(150)
(151) . ( FROL2 GD\
(152)
(153)
(154)
(155)
(156)
(157). & MHMXQL GD\
(158)
(159) [
(160) [
(161)
(162) [
(163) . . (DFKGDWDSRLQWLQGLFDWHVWKHPHDQRISRVLWLYHDQLPDOV6KRZQEHWZHHQEUDFNHWVLVWKHQXPEHURI SRVLWLYHDQLPDOVRXWRIWKUHH'DVK
(164) DOODQLPDOVEHORZGHWHFWLRQOHYHO FIXJ
(165). +DHPDWRORJ\. 'HWDLOHGLQIRUPDWLRQRQWKHUHVXOWVRIKDHPDWRORJLFDODQDO\VLVLVJLYHQLQ)LJXUH)LJXUH 7DEOHVKRZVDVXPPDU\RIVLJQLILFDQWFKDQJHVLQWKHEORRGSDUDPHWHUV:LWKRQHRUPRUH SDWKRJHQVWKHUHZDVDVLJQLILFDQWGHFUHDVHLQWKHEDVHOLQHOHYHORIVHYHUDOSDUDPHWHUVUHODWHG WRUHGEORRGFHOOV +JE0&+0&+&DQG5': QRWHDQLQFUHDVHZLWK& MHMXQLIRU WKH ODWWHUSDUDPHWHU
(166) 7KLVPLJKWLQGLFDWHDQDHPLDLQGXFHGE\H[SHULPHQWDOFRQGLWLRQVZKLFK FRXOGEHUHODWHGWRWKHUHSHDWHGVDPSOLQJRIEORRGIURPWKHDQLPDOV DWGD\±DWGD\RU DQG DWGD\RUDWDXWRSV\
(167) 7KHEDVHOLQHOHYHORIVHYHUDOZKLWHEORRGSDUDPHWHUV :%& PRQRF\WHVO\PSKRF\WHVEDVRSKLOV/8&V
(168) DOVRGHFUHDVHGZLWKRQHRUPRUHSDWKRJHQV$ GRVHGHSHQGHQWLQFUHDVHLQWKHQXPEHURIFHOOVLQYROYHGLQLQIODPPDWRU\UHDFWLRQV QHXWURSKLOVDQGPRQRF\WHV
(169) ZDVREVHUYHGLQDQLPDOVH[SRVHGWR6 (QWHULWLGLVLQGLFDWLQJ VHYHUHV\VWHPLFHIIHFWVRIWKHVHEDFWHULD,QWKH6 (QWHULWLGLVH[SRVHGDQLPDOVWKHUHZDVDOVR D GRVHUHODWHGGHFUHDVHRI0&9DQG0&+7KLVPLJKWLQGLFDWHWKDWWKHRYHUDOOUHGXFWLRQRI KDHPRJORELQE\H[SHULPHQWDOFRQGLWLRQVLVHQKDQFHGE\H[SRVXUHWR6 (QWHULWLGLV SRVVLEO\ E\OHVVHIILFLHQWVXSSOHPHQWDWLRQ
(170) 1RV\VWHPLFHIIHFWVZHUHREVHUYHGDIWHUH[SRVXUHWR( FROL2RU& MHMXQL 7DEOH&KDQJHVLQKDHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWRWKUHH HQWHURSDWKRJHQLFEDFWHULD 5HGEORRGFHOOV +DHPRJORELQ +&7 0&9 0&+ 0&+& 5': +': 3ODWHOHWV 039 :KLWHEORRGFHOOV 1HXWURSKLOV /\PSKRF\WHV 0RQRF\WHV (RVLQRSKLOV %DVRSKLOV /DUJHXQVWDLQHGFHOOV OXF
(171) Æ 6LJQLILFDQWLQFUHDVHLQEDVHOLQHOHYHO !
(172) É 6LJQLILFDQWGRVHGHSHQGHQWLQFUHDVH !
(173). 6(QWHULWLGLV GD\
(174). (FROL2 GD\
(175). Ç Ë ÇË Ç Ç Æ Ç ÇÉ É Ë ÇÉ. &MHMXQL GD\
(176) Ç. Ç Ç Ç Æ Ç Æ Ç Ç. Ç Æ Ç Ç. ÇÉ ÇË ÇÉ Ç ÇÉ Ç ÇÉ Ç 6LJQLILFDQWGHFUHDVHLQEDVHOLQHOHYHO
(177) Ë 6LJQLILFDQWGRVHGHSHQGHQWGHFUHDVH
(178).
(179) SDJHRI. PPRO O. 5,90UHSRUW. +JE. OO . IO . . . . . 'RVH. 0&9. . . . . . 'RVH. 5':. . . IO . . [ O [ O . . . . 'RVH. . . . . 'RVH. . . . 'RVH. EDVR DEV. . . . . . . . . . . IPRO . . PPRO O . . . . 'RVH. . . . 'RVH. 0&+. . . . 'RVH. . . PPRO O . 5%&. . . . . . . . . . . . . 'RVH. 0&+&. . [ O. +':. . 'RVH. 3OW. . . . . 'RVH. . . . [ O . :%&. . 'RVH. QHXW DEV. . . [ O . O\PSK DEV. . . [ O. 039. [ O. +FW. [ O . . . . 'RVH. PRQR DEV. . . . . . . 'RVH. [ O . . . 'RVH. HRV DEV. . . 'RVH. OXF DEV. . . 'RVH. )LJXUH+DHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDODQLPDOV'RVH ORJGRVHSHU DQLPDO FRQWURO :*
(180) Å EHIRUHH[SRVXUH GD\±
(181) × DIWHUH[SRVXUH GD\
(182) .
(183) 5,90UHSRUW. PPRO O. +JE. OO. . . IO . SDJHRI. . . . 'RVH. 0&9. . . . . . . IPRO . . 'RVH. . . . . 'RVH. 0&+. . PPRO O . 5':. [ O . +FW. . . . 'RVH. +':. . . . IO . . . . 'RVH. . . [ O. 039. . . . 'RVH. . . . . [ O. . . . . [ O . . . 'RVH. . . . 'RVH. . . . . . . . . PPRO O . . . . 'RVH. . . . . . . . . . . . . [ O . 'RVH. 3OW. . . 'RVH. QHXW DEV. . 'RVH. 0&+&. [ O . . . . 'RVH. HRV DEV. . . 'RVH. OXF DEV. . 'RVH. PRQR DEV. [ O. EDVR DEV. . . [ O. O\PSK DEV. . . . [ O. :%&. . 'RVH. 5%&. . . 'RVH. )LJXUH+DHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR(FROL2 (DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDODQLPDOV'RVH ORJGRVHSHU DQLPDO FRQWURO :*
(184) Å EHIRUHH[SRVXUH GD\±
(185) × DIWHUH[SRVXUH GD\
(186) .
(187) SDJHRI. PPRO O . . +JE. . IO . 5,90UHSRUW. . OO. . . 'RVH. 0&9. . . . . . 'RVH. 5':. . IO . . . . 'RVH. . . . [ O O\PSK DEV [ O . . . IPRO . . . 'RVH. . . . 'RVH. . . . . . 'RVH. +':. . . . 'RVH. . . [ O . 5%&. . PPRO O . . [ O . . . . 'RVH. 'RVH. . . . . . . . . 'RVH. 3OW. . . 'RVH. QHXW DEV. . 'RVH. 0&+&. [ O. [ O PRQR DEV . . . . [ O HRV DEV . . . 'RVH. 'RVH. OXF DEV. . 'RVH. :%&. [ O. . 0&+. PPRO O . 'RVH. EDVR DEV. . . [ O . 039. . . +FW. . . . . 'RVH. )LJXUH+DHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR&MHMXQL (DFK V\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDODQLPDOV'RVH ORJGRVHSHU DQLPDO FRQWURO :*
(188) Å EHIRUHH[SRVXUH GD\±
(189) × DIWHUH[SRVXUH GD\
(190) .
(191) RIVM report 284550 007. page 19 of 34. 2.3.5 Pathology In rats, infected with E.Coli O157 or C. jejuni, no gross or microscopical abnormalities were found. In rats, infected with S. Enteritidis, bronchopneumonia was detected in the highest dose group. Gross gastro-intestinal abnormalities were limited: little gastro-intestinal content in the highest dose S. Enteritidis dose group, and little gastric content in the second highest group, reflecting reduced food intake; enlarged Peyers’ patches and mesenteric lymph nodes and thickening of the caecal wall in both groups. Microscopically, in many animals (also in controls) isolated villi (< 1 per 100) distended by mononuclear cells (sometimes purely plasma cells) were seen, especially in the duodenum and jejunum. This was left aside when assessing the “itis”-score. The histopathological findings of animals exposed to S. Enteritidis are summarised in Table 5. No abnormalities were seen in the duodenum and distal colon. In all but one animal in the two highest dose groups predominantly mononuclear inflammatory infiltration of the villi in the ileum was present, while the jejunum was generally normal. In one animal however the situation was the opposite. In order to illustrate these peculiarities, individual histopathological data are given in Appendix 2. Moderate to marked infiltration of the small intestinal mucosa (‘villitis’) was accompanied with a decreased villus-crypt ratio. Microgranulomas were seen mainly in Peyers’ patches, in one animal associated with local peritonitis, and also in the lamina propria of animals in the two highest dose groups, as well as in one animal in the intermediate group. In the large intestine microgranulomas were limited to the GALT. Inflammation reached a higher grade of severity in the large intestine, especially in the caecum of the 3 highest dose groups. In the two highest groups diffuse flattening of superficial caecal epithelium, massive erosions and local ulcerations and decreased PAS-positivity were seen. In association almost diffusely, very marked, mixed inflammatory infiltration was present in the mucosa, near ulcers also in the submucosa, accompanied with marked oedema. Caecal lesions in the intermediate group locally reached equal severity, but were less extensive. Infiltration was also present in the proximal but not in the distal part of the colon. Table 5. Histopathologic evaluation of enteritis (D: duodenum, J: jejunum, I: ileum, C: caecum, C1: proximal colon, C2: distal colon), of adult, male WU rats infected with different doses of S. Enteritidis. Severity class. Dose (log10) 9.4 7.6 5.5 3.7 1.2 Control. D -. J ++ -. I + + + -. Severity classes of enteritis: - no, + slight, ++ moderate, +++marked. C +++ +++ +++ -. C1 ++ ++ + -. C2 -.
(192) page 20 of 34. RIVM report 284550 007. Figure 7. Left panel: destruction of crypts and diffuse epithelial flattening and erosion, massive mixed infiltration in (sub)mucosa of the caecum of a rat, infected with S. Enteritidis (highest dose group). Right panel: normal control. Note high columnar epithelium, regular crypts and low cellularity of stroma. HE, bar is 40 µm.. 2.4 Conclusion Exposure by intragastric gavage of adult, male WU rats to three enteropathogenic bacteria results in dose-dependent colonisation of the gastro-intestinal tract. C. jejuni was the most infectious of three species tested, with 2/3 animals infected at a dose of only 70 cfu per animal. E. coli O157 infected 2/3 animals at a dose of 5,700 cfu per animal, but 0/3 animals were infected at a dose of 50 cfu per animal. S. Enteritidis was least infectious, a dose of 330,000 cfu per animal but not lower doses infected 3/3 animals. For all bacteria, faecal counts increased with dose. For C. jejuni, peak values were obtained more rapidly at higher doses. S. Enteritidis was highly invasive, resulting in bacteraemia, pneumonia and reduced growth in the highest dose group. Macroscopically, limited gastro-intestinal abnormalities were observed only in the S. Enteritidis treated animals. There were only minor effects on body temperature and faecal moisture content. Microscopically, dose-dependent inflammation of the villi in the ileum, but not the duodenum, and granuloma in the Peyers’ patches of the jejunum and ileum was demonstrated in animals exposed to S. Enteritidis. Severe, dose-dependent inflammation in the caecum and in the proximal colon was also observed in these animals. E. coli O157 and C. jejuni did not induce remarkable pathological changes. Experimental conditions (probably repeated blood sampling) induced anaemia in all animals, including control groups. In the highest dose groups of S. Enteritidis, anaemia was significantly more pronounced than in other groups. White blood cells (notably neutrophils) increased markedly in animals exposed to S. Enteritidis, with a highly significant doseresponse relation..
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
(194) 1D+&2 . . . 5HVXOWV. ,QRFXOXPFXOWXUHV. 7DEOHVKRZVWKHSODWHFRXQWVRIGLIIHUHQWGLOXWLRQVRIWKHLQRFXOXPFXOWXUH)RUFRQYHQLHQFH LQODWHUVHFWLRQVHDFKGRVHJURXSLVDVVLJQHGDOHWWHUFRGH7KHLQWHQGHGUDQJHRIFIX SHUDQLPDOZDVREWDLQHGLQWKHILUVWFRKRUWEXWLQWKHVHFRQGFRKRUWWKHGRVHVZHUHDERXW IROGKLJKHU 7DEOH0LFURELRORJLFDODQDO\VLV FRORQ\FRXQWSHUPO
(195) RI&MHMXQLLQLQRFXOXPFXOWXUHV. 'RVHJURXS +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH 6HFRQGORZHVW /RZHVW &RQWURO :*
(196). FRKRUW . &OLQLFDODQGPDFURVFRSLFREVHUYDWLRQV. FRKRUW . 1RFOLQLFDOVLJQVRILQIHFWLRQRUGLVHDVHZHUHREVHUYHG$WQHFURSV\QRDEQRUPDOLWLHVZHUH GHWHFWHG. . 7KLVH[SHULPHQWLVUHJLVWHUHGDV$$3 .
(197) SDJHRI. 5,90UHSRUW. 0LFURELRORJ\. 2QO\RQHRIDQLPDOVLQIHFWHGZLWK& MHMXQLLQ GRVHVEHWZHHQDQGSHUDQLPDOZDV LQIHFWHGDVLQGLFDWHGE\IDHFDOH[FUHWLRQ 7DEOH
(198) ,QWKLVDQLPDOIDHFDOFRXQWVYDULHG EHWZHHQ DQG FIXJ . . 7DEOH)DHFDOH[FUHWLRQRI&MHMXQLE\DGXOWPDOH:UDWVDIWHURUDOH[SRVXUHWRGLIIHUHQWGRVHV. 'RVHJURXS +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH 6HFRQGORZHVW /RZHVW &RQWURO :*
(199). FRKRUW . FRKRUW . ,QYLHZRIWKHVHUHVXOWVLWZDVGHFLGHGQRWWRUHSRUWKDHPDWRORJLFDOGDWD+LVWRSDWKRORJLFDO H[DPLQDWLRQVZHUHQRWSHUIRUPHG. &RQFOXVLRQ 7KHUHSURGXFLELOLW\RIWKHUDWPRGHOZLWK& MHMXQLDV DWHVWRUJDQLVPLVYHU\SRRU1R REYLRXVH[SODQDWLRQZDVIRXQGZKHQUHYLHZLQJWKHH[SHULPHQWDOFRQGLWLRQV WKHUDWVZHUH REWDLQHGIURPWKHVDPHEUHHGLQJFRORQ\DQGZHUHFRORQLVHGZLWKWKHVDPH&5)IORUD7KHUH ZHUH PLQRUGLIIHUHQFHVLQWKHSHULRGRIIDVWLQJ KLQWKLVH[SHULPHQWYVKLQWKH SUHYLRXV
(200) EXWWKLVZRXOGEHH[SHFWHGWRLQFUHDVHWKHDQLPDOV¶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¶GLHW$WWKH5,90UDWVDUHIHGDFRQYHQWLRQDOGLHWWKHFRPSRVLWLRQRIZKLFKLV NQRZQWRYDU\ZLWKWKH VHDVRQDO
(201) DYDLODELOLW\RILQJUHGLHQWV/RHVEHUJ
(202) GHPRQVWUDWHG WKDWHYHQPLQRUFKDQJHVLQIRRGSHOOHWVWKDWDUHQRWQRUPDOO\UHSRUWHGWRVFLHQWLVWVPD\ LQGXFHVLJQLILFDQWDOWHUDWLRQVLQWKHLPPXQHV\VWHPRIURGHQWVDQGWKXVFRXOGDOVRLQIOXHQFH WKHUDWV¶VXVFHSWLELOLW\WREDFWHULDOLQIHFWLRQ,QWKDWFDVHLWZRXOGEHQHFHVVDU\WRFKDQJHWRD V\QWKHWLFGLHW.
(203) 5,90UHSRUW. SDJHRI. 5HSURGXFLELOLW\RILQIHFWLRQZLWK6(QWHULWLGLV ,QWURGXFWLRQ. 7KHSRRUDQGSUHVHQWO\XQH[SODLQHGUHSURGXFLELOLW\RILQIHFWLRQZLWK& MHMXQL VHH&KDSWHU
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±&IRUWKH'7+UHDFWLRQ VHH
(205) +LVWRSDWKRORJLFDO H[DPLQDWLRQZDVFRQILQHGWRWKHFDHFXP. 'HOD\HGW\SHK\SHUVHQVLWLYLW\UHDFWLRQ '7+
(206). )LYHGD\VDIWHURUDOLQIHFWLRQZLWK6 (QWHULWLGLVWKHWKLFNQHVVRIERWKHDUVRIHDFKDQLPDOLQ HDFK JURXSZDVDVVHVVHGLQGXSOLFDWHXVLQJDQHQJLQHHULQJPLFURPHWHU 0LWXWR\R 9HHQHQGDDO7KH1HWKHUODQGV
(207) )RUWKLVSXUSRVHWKHDQLPDOVZHUHDQDHVWKHWLVHGE\ LQWUDSHULWRQHDOLQMHFWLRQRImO RID.5$VROXWLRQ DFRFNWDLOFRQVLVWLQJRIPONHWDODU PJPO3DUNH'DYLV6SDLQ
(208) POURPSXQ PJPO%D\HU/HYHUNXVHQ)5*
(209) DQGPORI DWURSLQ PJPO23*8WUHFKW7KH1HWKHUODQGV
(210) 'LUHFWO\DIWHUWKHHDUPHDVXUHPHQWVmO RIWKHKHDWNLOOHGVXVSHQVLRQRI6(QWHULWLGLV DSSUR[LPDWHO\[ FIXPO
(211) ZDV VXEFXWDQHRXVO\LQMHFWHGLQWRWKHSLQQDHRIHDFKHDURIHDFKUDW DOVRLQWKHFRQWURO:* JURXS
(212) 7KHLQFUHDVHLQHDUWKLFNQHVVZDVDVVHVVHGKUVDIWHUFKDOOHQJHXQGHUHWKHU DQDHVWKHVLD7KHGLIIHUHQFHEHWZHHQHDUWKLFNQHVVSULRUWRDQGKUVDIWHULQMHFWLRQZDV FDOFXODWHGDQGUHIOHFWVWKH'7+UHVSRQVHDYDOLGSDUDPHWHUIRU7FHOOGHSHQGHQW LQYLYR
(213) LPPXQLW\WR6 (QWHULWLGLV7KHVZHOOLQJLQFRQWURODQLPDOVUHIOHFWWKHEDFNJURXQGVZHOOLQJ UHVSRQVHLQGXFHGE\WKHHDULQMHFWLRQRI6 (QWHULWLGLVLWVHOI . 5HVXOWV ,QRFXOXPFXOWXUHV. 7DEOHVKRZVWKHSODWHFRXQWVRIGLIIHUHQWGLOXWLRQVRIWKHLQRFXOXPFXOWXUHDJDLQDVVLJQHGD OHWWHUFRGHIRUHDFKJURXS1RWHWKDWDOOGRVHVZHUHDSSUR[LPDWHO\IROGORZHUWKDQLQWKH SUHYLRXVH[SHULPHQW 7DEOH
(214) %\PLVWDNHWKHLQRFXOXPRIWKHORZGRVHJURXSZDVSODWHG RQWKHZURQJPHGLXPKHQFHWKHGRVHLVQRWNQRZQGLUHFWO\,QWKHWDEOHDQHVWLPDWHGUDQJH LVJLYHQRQWKHEDVLVRIWKHFRXQWVLQWKHKLJKHUGRVHJURXSVDQGWKHGLOXWLRQUDWLR. . 7KLVH[SHULPHQWZDVUHJLVWHUHGDV$$3 .
(215) SDJHRI. 5,90UHSRUW. 7DEOH0LFURELRORJLFDODQDO\VLV FRORQ\FRXQWSHUPOORJLQEUDFNHWV
(216) RILQRFXOXPFXOWXUHV. 'RVHJURXS +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH /RZ &RQWURO :*
(217). 6 (QWHULWLGLV [
(218) [
(219) [
(220) [
(221) [
(222) . . . . ([WUDSRODWHGIURPKLJKHUGRVHJURXSVVHHWH[W. &OLQLFDOREVHUYDWLRQV. 7KHDQLPDOVLQIHFWHGZLWKWKHWZRKLJKHVWGRVHVVKRZHGVHYHUHLOOQHVVIURPGD\DIWHU LQIHFWLRQRQZDUG7KHDQLPDOVZHUHVNLQQ\ZHDNFROGKDGDUXIIOHGIXUUHGFUXVWVDURXQG H\HVDQGQRVHWLSVDQGLQFUHDVHGDPRXQWVRIQDVDOIOXLG6HYHUHV\VWHPLFLOOQHVVIURPGD\ RQZDUGVRFFXUUHGLQUDWVLQWKHKLJKHVWGRVHJURXSWKHVHZHUHVDFULILFHGDQGQHFURSVLHG DW GD\WKHRWKHUUDWVRQGD\DVSODQQHG,QWKHKLJKHVWGRVHJURXSDORVVRIPHDQERG\ ZHLJKWIURPGD\RQZDUGVZDVQRWHG VHH)LJXUH
(223) *UDP . &WUO. . . . . . . . . 'D\. . )LJXUH%RG\ZHLJKWVRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV(DFKV\PERO LQGLFDWHVWKHPHDQRILQGLYLGXDODQLPDOV/HJHQG ORJGRVHSHUDQLPDO&WUO FRQWURO :*
(224) . 7KHFDHFXPZHLJKWVLQDOODQLPDOVZHUHOHVVWKDQRIWKHWRWDOERG\ZHLJKWLQGLFDWLQJQR PDMRUDEQRUPDOLW\LQWKHUDW¶VLQWHVWLQDOPLFURIORUD&RPSDUHGWRWKHFRQWUROJURXSQR VLJQLILFDQWGLIIHUHQFHLQFDHFXPWRERG\ZHLJKWUDWLRZDVIRXQGLQDOOJURXSVLQIHFWHGZLWK6 (QWHULWLGLVH[FHSWWKHVHFRQGKLJKHVWGRVHJURXS VHH)LJXUH
(225) 3HUFHQW . . . . . . 'RVH. )LJXUH &DHFXPWRERG\ZHLJKWUDWLRRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRIILYHLQGLYLGXDOV WKUHHIRUGRVH
(226) 'RVH ORJGRVHSHUDQLPDO FRQWURO :*
(227) .
(228) 5,90UHSRUW. SDJHRI. 7KHUHZDVDGRVHGHSHQGHQWGHFUHDVHRIERG\WHPSHUDWXUHRIWKHLQIHFWHGDQLPDOVIRU PHDVXUHPHQWVWDNHQLQWKHDIWHUQRRQ%HFDXVHRIUHODWLYHO\KLJKLQWHULQGLYLGXDOYDULDELOLW\ WKHGLIIHUHQFHRQO\UHDFKHGVWDWLVWLFDOVLJQLILFDQFHIRUWKHVHFRQGKLJKHVWGRVHJURXS WZR VLGHGWWHVWRIH[SRVHGDQLPDOVYHUVXVFRQWUROVVHH)LJXUH
(229) EXWQRWIRUWKHKLJKHVWGRVH JURXSHYHQWKRXJKWKHPHDQWHPSHUDWXUHZDVFRQVLGHUDEO\ORZHU7HPSHUDWXUH PHDVXUHPHQWVWDNHQGXULQJWKHPRUQLQJGLGQRWVKRZDQ\GRVHGHSHQGHQWGLIIHUHQFHV7KLV PD\EHUHODWHGWRWKHIDFWWKDWUDWVDUHQLJKWIHHGHUVDQGFDQPDLQWDLQWKHLUERG\WHPSHUDWXUH E\SK\VLFDODFWLYLW\GHVSLWHWKHHIIHFWRILQIHFWLRQ'XULQJGD\WLPHWKHDQLPDOVVOHHSDQGD GHFUHDVHLQERG\WHPSHUDWXUHVLVQRWFRPSHQVDWHGE\SK\VLFDODFWLYLW\ & . & . . . . . . . . . . . . . . . 'RVH. . . . . . 'RVH. )LJXUH%RG\WHPSHUDWXUH DIWHUQRRQPHDVXUHPHQWV
(230) RIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR 6(QWHULWLGLV(DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRIILYHLQGLYLGXDOV'RVH ORJ GRVHSHUDQLPDO FRQWURO :*
(231) /HIWSDQHO GD\± ULJKWSDQHO GD\. ,QWKHWZRKLJKHVWGRVHJURXSVVLJQLILFDQWULVHVLQIDHFDOPRLVWXUHFRQWHQWZHUHREVHUYHG RYHUGD\V GD\±WRGD\VHH)LJXUH
(232) 0RLVWXUH. . &WUO . . . . . . . . 'D\. . )LJXUH0RLVWXUHFRQWHQWRIWKHIDHFHVRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQRIILYHLQGLYLGXDODQLPDOV/HJHQG ORJGRVHSHUDQLPDO&WUO FRQWURO :*
(233) . 0LFURELRORJ\. )LJXUHVKRZVWKHIDHFDOH[FUHWLRQRISDWKRJHQVLQUHODWLRQWRGRVHDQGWLPHDIWHU LQRFXODWLRQ$VLQWKHSUHYLRXVH[SHULPHQWH[SRVXUHWR6 (QWHULWLGLVUHVXOWHGLQDVKDUS LQFUHDVHRIIDHFDOFRXQWVIROORZHGE\DWUDQVLHQWGHFUHDVHDQGVXEVHTXHQWLQFUHDVH(YHQ WKRXJKWKHGRVHVZHUHWHQIROGORZHUWKDQLQWKHSUHYLRXVH[SHULPHQWWKHH[FUHWLRQSDWWHUQV ZHUH YHU\VLPLODU1R(FROL:*ZDVGHWHFWHGLQDQ\RIWKHIDHFDOVDPSOHVEORRGRUO\PSK QRGHV.
(234) SDJHRI. 5,90UHSRUW. /RJ FIX . &WUO . . . . . . 'D\. . )LJXUH&RORQ\FRXQWVLQWKHIDHFHVRIDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQRILQGLYLGXDODQLPDOV/HJHQG ORJ GRVHSHUDQLPDO&WUO FRQWURO :*
(235) . ,IDJDLQWKHFULWHULRQIRULQIHFWLRQLVGHILQHGDVDIDHFDOFRXQW!JRQDWOHDVWWZRGD\V WKHGRVHUHVSRQVHGDWDDVVKRZQLQ7DEOH DUHREWDLQHG,QIHFWLRQZDVDJDLQREVHUYHGLQWKH WKUHHKLJKHVWGRVHJURXSVKHQFHGRZQWRDGRVHRI[ FIXSHUDQLPDO . 7DEOH ,QIHFWLRQ GHILQHGE\IDHFDOH[FUHWLRQ
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
(237) LQPHVHQWHULFO\PSKQRGHVDQGVSOHHQRIDGXOWPDOH:8UDWV DIWHURUDOH[SRVXUHWRGLIIHUHQWGRVHV. 'RVHJURXS. +LJKHVW 6HFRQGKLJKHVW ,QWHUPHGLDWH /RZ. 0HVHQWHULF O\PSKQRGHV [
(238) [
(239) Ü
(240) Ü
(241) . 6SOHHQ. [ [ [ [. .
(242)
(243)
(244)
(245). (DFKGDWDSRLQWLQGLFDWHVWKHPHDQRISRVLWLYHDQLPDOV6KRZQLQEUDFNHWVLV WKHQXPEHURISRVLWLYHDQLPDOVRXWRIILYH $VWHULVN Ü
(246) LQWHUIHUHQFHE\EDFNJURXQGIORUD.
(247) 5,90UHSRUW. SDJHRI. 'HOD\HGW\SHK\SHUVHQVLWLYLW\UHDFWLRQ '7+
(248). ,QDOOLQIHFWHGDQLPDOVDVLJQLILFDQWVSHFLILFGHOD\HGW\SHK\SHUVHQVLWLYLW\UHVSRQVHZDV GHWHFWDEOHDVFRPSDUHGWRWKHEDFNJURXQGVZHOOLQJUHVSRQVHLQGXFHGE\6DOPRQHOOD LQMHFWLRQVLQWKHHDUVRIFRQWUROUDWV VHH)LJXUH
(249) 7KLVLQGLFDWHVDVLJQLILFDQWDQGGRVH UHODWHG V\VWHPLF7FHOOGHSHQGHQWLPPXQHUHVSRQVHWRWKH6 (QWHULWLGLV(YHQLQWKHORZHVW GRVHJURXSDVLJQLILFDQWUHVSRQVHZDVGHWHFWDEOHZKLFKLVFRQVLVWHQWZLWKWKHSUHVHQFHRI6 (QWHULWLGLVLQWKHVSOHHQ PP . . . . . 'RVH. )LJXUH'7+UHDFWLRQLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDODQLPDOV LQWKH9+JURXS
(250) 'RVH ORJGRVHSHUDQLPDO FRQWURO :*
(251) .
(252) SDJHRI. 5,90UHSRUW. +DHPDWRORJ\. 'HWDLOHGLQIRUPDWLRQRQWKHUHVXOWVRIKDHPDWRORJLFDODQDO\VLVLVJLYHQLQ)LJXUH7DEOH VKRZV DVXPPDU\RIVLJQLILFDQWFKDQJHVLQWKHEORRGSDUDPHWHUV 7DEOH&KDQJHVLQKDHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR 6 (QWHULWLGLV. 5HGEORRGFHOOV +DHPRJORELQ +&7 0&9 0&+ 0&+& 5': +': 3ODWHOHWV 039 :KLWHEORRGFHOOV 1HXWURSKLOV /\PSKRF\WHV 0RQRF\WHV (RVLQRSKLOV %DVRSKLOV /DUJHXQVWDLQHGFHOOV. Æ Æ Ç ÇË Ç Æ É É ÆÉ Ç É Ë É É. Æ 6LJQLILFDQWLQFUHDVHLQEDVHOLQHOHYHO !
(253) Ç 6LJQLILFDQWGHFUHDVHLQEDVHOLQHOHYHO !
(254) É 6LJQLILFDQWGRVHGHSHQGHQWLQFUHDVH !
(255) Ë 6LJQLILFDQWGRVHGHSHQGHQWGHFUHDVH
(256). $JDLQWKHUHZDVDVLJQLILFDQWGHFUHDVHLQWKHEDVHOLQHOHYHORIVRPHKDHPDWRORJLFDO SDUDPHWHUV 0&90&+0&+&DQGO\PSKRF\WHV
(257) 1RWHWKDWLQWKHSUHYLRXVH[SHULPHQW PRUH SDUDPHWHUVZHUHGHFUHDVHGDVFRPSDUHGWREDVHOLQH +JE5':039:%& PRQRF\WHVEDVRSKLOV/8&V
(258) ZKHUHDVLQWKLVH[SHULPHQWWKHUHZDVDOVRDQLQFUHDVHLQWKH EDVHOLQHOHYHORIVRPHSDUDPHWHUV 5%&+&7DQGQHXWURSKLOV
(259) 7KXVDQDHPLDLQGXFHGE\ H[SHULPHQWDOFRQGLWLRQVZDVOHVVSURQRXQFHGWKDQLQWKHSUHYLRXVH[SHULPHQW,QWKLV H[SHULPHQWEORRGZDVRQO\VDPSOHGDWGD\±EXWQRWDWGD\7KHLQFUHDVHLQQHXWURSKLOV PD\KDYHEHHQLQGXFHGE\LQRFXODWLRQRIKHDWNLOOHG6 (QWHULWLGLVIRUWKH'7+UHDFWLRQ $VLQWKHSUHYLRXVH[SHULPHQWDGRVHGHSHQGHQWLQFUHDVHLQWKHQXPEHURISKDJRF\WLFFHOOV LQYROYHGLQLQIODPPDWRU\UHDFWLRQV QHXWURSKLOVPRQRF\WHV
(260) ZDVREVHUYHG.
(261) 5,90UHSRUW. PPRO O . SDJH RI. OO. +JE. +FW. . . . IO. . . . 'RVH. 0&9. . . IPRO. . . . . . . . . . 0&+. . . . . . . . 'RVH. 5':. . . IO. . . . 'RVH. 039. . O. . . . 'RVH. O\PSK DEV. O . . . . . 'RVH. EDVR DEV. . . PPRO O . . . 'RVH. . . . . . . . . 'RVH. . . PPRO O . . . 'RVH. . . . . . . . . . . . . 'RVH. 'RVH. 3OW. . . . 'RVH. QHXW DEV. . . . O. PRQR DEV. 0&+&. O. :%&. . 5%&. O. . 'RVH. +':. O. . . . O. . . 'RVH. O . 'RVH. HRV DEV. . . O . . . . . . 'RVH. . . . 'RVH. OXF DEV. . . 'RVH. )LJXUH+DHPDWRORJLFDOSDUDPHWHUVLQDGXOWPDOH:8UDWVDIWHURUDOH[SRVXUHWR6(QWHULWLGLV (DFKV\PEROLQGLFDWHVWKHPHDQDQGVWDQGDUGGHYLDWLRQRILQGLYLGXDODQLPDOV'RVH ORJGRVHSHU DQLPDO FRQWURO :*
(262) Å EHIRUHH[SRVXUH GD\±
(263) × DIWHUH[SRVXUH GD\
(264) .
(265) SDJHRI. 5,90UHSRUW. 3DWKRORJ\. $ VXPPDU\RIWKHPDFURVFRSLFILQGLQJVLVJLYHQLQ7DEOH,QJHQHUDOWKHHIIHFWVZHUH PRUH SURQRXQFHGWKDQLQWKHSUHYLRXVH[SHULPHQW7KHUHZDVGRVHUHODWHGV\VWHPLFLOOQHVV QRWDEO\SQHXPRQLDDQGK\GURWKRUD[$OVRWKHUHZHUHOLPLWHGGRVHUHODWHGJDVWURLQWHVWLQDO DEQRUPDOLWLHV OLWWOHJDVWURLQWHVWLQDOFRQWHQWUHIOHFWLQJUHGXFHGIRRGLQWDNHDEQRUPDO FUXPEO\JDVWURLQWHVWLQDOFRQWHQW QRWVHHQLQSUHYLRXVVWXGLHV
(266) DQGRSDFLW\RIWKHFDHFDOZDOO 3H\HUV¶SDWFKHVLQWKHLOHXPDQGWKHPHVHQWHULFO\PSKQRGHVZHUHHQODUJHGLQWKHWZR KLJKHVWGRVHJURXSV7KHWZRUDWVWKDWZHUHNLOOHGRQGD\ZHUHWKHRQO\RQHVLQWKHLUJURXS ZLWKRXWREYLRXVW\SKOLWLV PDLQO\FU\SWK\SHUSODVLD
(267) EXWFDHFDO*$/7VKRZHG PLFURJUDQXORPDVLQERWK +LVWRSDWKRORJLFDOOHVLRQVLQWKHFDHFXP $SS
(268) ZHUHFRPSDUDEOHWRWKRVHVHHQLQWKH SUHYLRXVVWXG\
(269) EXWPRUHYDULDWLRQLQWKHVHYHULW\RIPXFRVDOOHVLRQVZDVREVHUYHGLQ WKHLQWHUPHGLDWHGRVHJURXS,QFDHFDDEQRUPDOPXFRVDSUHGRPLQDWHGDQGVXEPXFRVDO VRPHWLPHVWUDQVPXUDO
(270) LQIODPPDWRU\LQILOWUDWLRQZDVPDUNHGWRVHYHUH 7DEOH0DFURVFRSLFILQGLQJVLQ\RXQJDGXOW:8UDWVH[SRVHGWRGLIIHUHQWGRVHVRI6(QWHULWLGLV. 'RVHJURXS. +LJKHVW 6HFRQG KLJKHVW ,QWHUPHGLDWH /RZ &WUO :*
(271). 3QHXPRQLD. +\GURWKRUD[. . . . . 2SDFLW\ FDHFXP . /LWWOH*, FRQWHQWV . $EQRUPDO*, FRQWHQWV . &RQFOXVLRQ. 7KHGRVHUHVSRQVHUHODWLRQVKLSRIH[SRVXUHE\LQWUDJDVWULFJDYDJHRIDGXOWPDOH:8UDWVWR 6 (QWHULWLGLVDSSHDUVWREHZHOOUHSURGXFLEOH$VLQWKHSUHYLRXVH[SHULPHQWWKHDQLPDOV ZHUH VKHGGLQJWKHEDFWHULDLQWKHLUIDHFHVDIWHUH[SRVXUHWRLQWHUPHGLDWHWRKLJKGRVHV ! FIXDQLPDO
(272) $WDORZHUGRVH FIXDQLPDO
(273) QRIDHFDOH[FUHWLRQZDVGHWHFWHGZLWKLQWKH GD\SHULRGRIREVHUYDWLRQEXWVDOPRQHOODHFRXOGEHLVRODWHGIURPWKHVSOHHQDQGPHVHQWHULF O\PSKQRGHV)XUWKHUPRUHDOUHDG\DWWKLVGRVHWKHUHZDVDSRVLWLYH'7+UHDFWLRQ7KHVH ILQGLQJVXQGHUOLQHWKHIDFWWKDW6 (QWHULWLGLVLVKLJKO\LQYDVLYHLQURGHQWVDQGWKDWWKH LQWHVWLQDOWUDFWLVQRWWKHPDMRUVLWHRIPXOWLSOLFDWLRQ 3DWKRORJLFDOUHVXOWVFRQILUPREVHUYDWLRQVLQDSUHYLRXVVWXG\ *DUVVHQHWDO
(274) ZKLFK LQGLFDWHGWKDWWKHJDVWURLQWHVWLQDOWUDFW DOWKRXJKSRUWDORIHQWU\
(275) VKRZVUHODWLYHO\OLWWOH DEQRUPDOLWLHVLQDQLPDOVWKDWVXFFXPEDIWHURUDOLQRFXODWLRQZLWKYHU\KLJKGRVHVRI6 (QWHULWLGLV2QWKHRWKHUKDQGV\VWHPLFLOOQHVVZDVVHYHUHDVGHPRQVWUDWHGE\SQHXPRQLD DQG K\GURWKRUD[ .
(276) 5,90UHSRUW. SDJHRI. 5HIHUHQFHV *DUVVHQ-+DYHODDU$+'XIUHQQH-%.RHGDP0'H-RQJ:+7DNXPL.7HXQLV3)0 9DQ/HXVGHQ)09RV-*'RVHUHVSRQVHPRGHOVIRUJDVWURLQWHVWLQDOSDWKRJHQVLQDQ DQLPDO PRGHO,QWHULPUHVXOWV1DWLRQDO,QVWLWXWHRI3XEOLF+HDOWKDQGWKH(QYLURQPHQW %LOWKRYHQ 5HSRUWQR *XLQp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
(277) SDJHRI. $SSHQGL[0DLOLQJOLVW. 'LUHFWRU*HQHUDO5,90 6/HDFK&$053RUWRQ'RZQ8. 9/HFOHUF$)66$0DLVRQ$OIRUW)UDQFH -+XLVLQµ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
(278) 5,90UHSRUW. SDJHRI. $SSHQGL[,QGLYLGXDOKLVWRSDWKRORJLFDOGDWD 6(QWHULWLGLVH[SHULPHQW$$3 GRVH ORJ
(279). DQLPDO QU. GXRGHQXP YLOOLWLV. MHMXQXP YLOOLWLV. LOHXP YLOOLWLV. FDHFXP LQILOWUDWLRQ. FRORQ LQILOWUDWLRQ. FRORQ LQILOWUDWLRQ. < < < < < " " < < < " " " " " " j < j < < < < < < < < < < < < /HJHQG 6HH$SSHQGL[ 7KHYLOOXVFU\SWUDWLRLQWKHFRQWURODQLPDOVZDVHVWLPDWHGDWGXRGHQXPj MHMXQXP LOHXP . DXWRO\WLF &RQWURO.
(280) SDJHRI. 5,90UHSRUW. $SSHQGL[,QGLYLGXDOKLVWRSDWKRORJLFDOGDWD 6(QWHULWLGLVH[SHULPHQW$$3 'RVH ORJ
Afbeelding
GERELATEERDE DOCUMENTEN
These materials showed enhanced catalytic activity in relevant reactions compared to their counterpart catalyst prepared without scCO 2 or state-of-the-art
In general one can say that for good speech intelligibility a strong direct sound field is required, if possible, augmented with several early reflection (first 15 ms; at least
I) Determinanten en kenmerken van een stralingsincident. II) De gevolgen van een stralingsincident. III) De maatregelen die genomen kunnen/moeten worden om de nadelige gevolgen
Daarnaast is tussen 2004 en 2014 het aandeel van de bevolking dat gebruik maakt van zorg met verblijf bij 65- tot 80-jarigen en bij 80-plussers gedaald, maar niet bij mannen
Tabel 1: Toetswaarden voor nutriënten in grond- en oppervlaktewater (normen voor waterbeheer, 2000) Grondwater (mg/l) Oppervlaktewater (mg/l) Streefwaarde zandregio
Niet-geïnfecteerde partner(s) en huisgenoten van betrokkene komen in aanmerking voor bescherming tegen HBV-infectie middels vaccinatie. • Bij een gezondheidszorgwerker met
We observed that as compared to nonsmoking during pregnancy, quitting smoking in the first trimester is associated with the same risk of preterm birth and small size for
In the postmodern scene, panic signifies a twofold free-fall: the disappearance of ex- ternal standards of public conduct when the social itself becomes the
