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Állatorvostudományi Egyetem Doktori Iskola Nem Tuberkulotikus Mycobacteriumok Azonosítása És Tipizálása Phd Értekezés Dr. Rónai Zsuzsanna 2016

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Állatorvostudományi Egyetem Doktori Iskola Nem Tuberkulotikus Mycobacteriumok Azonosítása És Tipizálása Phd Értekezés Dr. Rónai Zsuzsanna 2016 Állatorvostudományi Egyetem Doktori Iskola Nem tuberkulotikus mycobacteriumok azonosítása és tipizálása PhD értekezés dr. Rónai Zsuzsanna 2016 Témavezető és témabizottsági tagok: ……………………………………..…. Dr. Dán Ádám Nemzeti Élelmiszerlánc-biztonsági Hivatal, Állat-egészségügyi Diagnosztikai Igazgatóság, Molekuláris Biológiai Laboratórium témavezető ……………………………………..…. Dr. Tuboly Sándor Állatorvostudományi Egyetem Járványtani és Mikrobiológiai Tanszék témabizottság tagja ……………………………………..…. Dr. Gyuranecz Miklós Magyar Tudományos Akadémia, Agrártudományi Kutatóközpont, Állatorvos-tudományi Kutatóintézet témabizottság tagja Készült 10 példányban. Ez a n. …. sz. példány. ……………………………………..…. dr. Rónai Zsuzsanna „Per aspera ad astra” -3- Tartalomjegyzék TARTALOMJEGYZÉK ...................................................................................................................................... 4 ÁBRÁK JEGYZÉKE ........................................................................................................................................... 5 TÁBLÁZATOK JEGYZÉKE .............................................................................................................................. 5 RÖVIDÍTÉSEK JEGYZÉKE .............................................................................................................................. 6 1. ÖSSZEFOGLALÁS .......................................................................................................................................... 9 2. SUMMARY ..................................................................................................................................................... 10 3. BEVEZETÉS ................................................................................................................................................... 11 4. IRODALMI ÁTTEKINTÉS .......................................................................................................................... 13 4.1. A MYCOBACTERIUM NEMZETSÉG ................................................................................................................ 13 4.2. A MYCOBACTERIUMOK JELLEMZŐI ............................................................................................................ 14 4.3. A MYCOBACTERIUMOK JELENTŐSÉGE, AZ OKOZOTT KÓRKÉPEK ................................................................ 17 4.4. A MYCOBACTERIUMOK EVOLÚCIÓJA ÉS CSOPORTOSÍTÁSA ........................................................................ 20 4.5. A MYCOBACTERIUMOK TENYÉSZTÉSE ....................................................................................................... 22 4.6. A MYCOBACTERIUMOK AZONOSÍTÁSA, GENOTIPIZÁLÁSA .......................................................................... 23 4.7. A MAGYARORSZÁGI ÁLLAT-EGÉSZSÉGÜGYI MYCOBACTERIUM DIAGNOSZTIKA .......................................... 26 4.8. CÉLKITŰZÉSEK .......................................................................................................................................... 28 5. ANYAG ÉS MÓDSZER ................................................................................................................................. 29 5.1. MINTAFELDOLGOZÁS, MYCOBACTERIUM TENYÉSZTÉS ............................................................................... 29 5.2. TÁPTALAJOK VIZSGÁLATA, ZIEHL−NEELSEN FESTÉS ................................................................................ 30 5.3. TENYÉSZETEK MORFOLÓGIAI ÉS NÖVEKEDÉSI SAJÁTOSSÁGAINAK VIZSGÁLATA, ÁTOLTÁSOK .................. 32 5.4. DNS KIVONÁS ........................................................................................................................................... 33 5.5. MYCOBACTERIUMOK AZONOSÍTÁSA I. − PCR MÓDSZEREK ADAPTÁLÁSA ................................................. 34 5.6. MYCOBACTERIUMOK AZONOSÍTÁSA II. − PCR MÓDSZERFEJLESZTÉS ........................................................ 37 5.7. SZEKVENÁLÁS, SZEKVENCIAELEMZÉS ....................................................................................................... 38 A 5.8. LSP 17 ÉS MIRU−VNTR ANALÍZIS ......................................................................................................... 39 5.9. FELHASZNÁLT TÖRZSEK ............................................................................................................................ 40 6. EREDMÉNYEK.............................................................................................................................................. 41 6.1. MYCOBACTERIUM AVIUM SUBSP. AVIUM (MAA) ......................................................................................... 42 6.2. MYCOBACTERIUM AVIUM SUBSP. SILVATICUM (MAS) .................................................................................. 43 6.3. MYCOBACTERIUM AVIUM SUBSP. HOMINISSUIS (MAH) ................................................................................ 46 6.4. MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS (MAP) ...................................................................... 47 6.5. EGYÉB, NEM M. AVIUM NTM-OK KIMUTATÁSA ÉS AZONOSÍTÁSA .............................................................. 49 6.6. VEGYES FERTŐZÉSEK ................................................................................................................................ 55 6.7. M. AVIUM ALFAJOK GENOTIPIZÁLÁSA ....................................................................................................... 55 6.8. TÖRZSGYŰJTEMÉNY .................................................................................................................................. 61 7. MEGBESZÉLÉS ............................................................................................................................................. 62 8. ÚJ TUDOMÁNYOS EREDMÉNYEK .......................................................................................................... 75 9. IRODALOMJEGYZÉK ................................................................................................................................. 77 10. A DOKTORI KUTATÁS EREDMÉNYEINEK KÖZLÉSEI ................................................................. 110 11. A DOKTORI KUTATÁS TÉMÁJÁHOZ NEM KAPCSOLÓDÓ TUDOMÁNYOS KÖZLEMÉNYEK ............................................................................................................................................................................ 115 12. MELLÉKLETEK ....................................................................................................................................... 119 13. KÖSZÖNETNYILVÁNÍTÁS .................................................................................................................... 129 -4- Ábrák jegyzéke 1. ÁBRA: ROBERT KOCH ................................................................................................................................. 13 2. ÁBRA: MYCOBACTERIUM TUBERCULOSIS TELEPEK LÖWENSTEIN−JENSEN TÁPTALAJON ....... 15 3. ÁBRA: AZ AMERIKAI VÖRÖSKERESZT TUBERKULÓZIS KAMPÁNYÁNAK PLAKÁTJA .............. 17 4. ÁBRA: GENOTYPE MYCOBACTERIUM CM ÉS MTBC KITEK EREDMÉNYEI ................................... 25 5. ÁBRA: A GÜMŐKÓRMENTESSÉGET HÍRDETŐ TÁBLA ........................................................................ 27 6. ÁBRA: ZIEHL−NEELSEN SZERINT FESTETT BÉLNYÁLKAHÁRTYA KAPARÉK ............................. 31 7. ÁBRA: MYCOBACTERIUM TÖRZSEK PCR IDENTIFIKÁLÁSA ............................................................... 35 8. ÁBRA: MULTIPLEX MYCOBACTERIUM PCR .......................................................................................... 36 9. ÁBRA: MAP IDENTIFIKÁLÓ ÉS TIPIZÁLÓ PCR RENDSZEREK ............................................................ 36 10. ÁBRA: IS901-IS1245 PCR ............................................................................................................................ 36 11. ÁBRA: LSPA17 PCR ...................................................................................................................................... 39 12. ÁBRA: MYCOBACTERIUM AVIUM SUBSP. AVIUM OKOZTA ELVÁLTOZÁSOK ............................... 42 13. ÁBRA: MYCOBACTERIUM AVIUM SUBSP. SILVATICUM TÖRZS 7H11 TALAJON ............................ 43 14. ÁBRA: OLVADÁSI GÖRBÉK A DUPLEX MM-HRM VALÓS IDEJŰ PCR RENDSZERBEN .............. 45 15. ÁBRA: NORMALIZÁLT GÖRBE ................................................................................................................ 45 16. ÁBRA: MEGNAGYOBBODOTT PEYER-PLAKKOK KUTYA VÉKONYBELÉBEN ............................. 47 17. ÁBRA: JUH TÍPUSÚ (S, I-ES) MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS TÖRZS J TALAJON ........................................................................................................................................ 47 18. ÁBRA: 34 REPREZENTATÍV NEM M. AVIUM NTM TÖRZS FILOGENETIKAI FÁJA ........................ 54 19. ÁBRA: M. AVIUM GENOTÍPUSOK FILOGENETIKAI FÁJA ................................................................... 60 Táblázatok jegyzéke 1. TÁBLÁZAT: KÜLÖNBÖZŐ KÓRFORMÁKBÓL IZOLÁLT MYCOBACTERIUM FAJOK ...................... 20 2. TÁBLÁZAT: MYCOBACTERIUM FAJOK AZONOSÍTÁSÁHOZ ALKALMAZOTT GÉNEK .................. 25 3. TÁBLÁZAT: 2006 ÉS 2015 KÖZÖTT AZONOSÍTOTT MYCOBACTERIUM TÖRZSEK ......................... 41 4. TÁBLÁZAT: 2006 ÉS 2015 KÖZÖTT AZONOSÍTOTT NTM-OK ÁLLATFAJONKÉNT ......................... 41 5. TÁBLÁZAT: 2006 ÉS 2015 KÖZÖTT AZONOSÍTOTT MAA TÖRZSEK ADATAI ................................
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