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Állatorvostudományi Egyetem Állatorvostudományi Doktori Iskola Állatorvostudományi Egyetem Állatorvostudományi Doktori Iskola Vírusok okozta komplex oktanú enterális megbetegedések sertésekben, különös tekintettel a coronavírusokra PhD értekezés dr. Valkó Anna 2019 Témavezetők: Prof. Tuboly Tamás† Állatorvostudományi Egyetem Járványtani és Mikrobiológiai Tanszék témavezető dr. Cságola Attila Ceva-Phylaxia Zrt. témavezető Készült 8 példányban. Ez a n. … sz. példány. …………………………………… dr. Valkó Anna 2 TARTALOMJEGYZÉK ÁBRÁK JEGYZÉKE ............................................................................................................. 5 TÁBLÁZATOK JEGYZÉKE .................................................................................................. 6 RÖVIDÍTÉSEK JEGYZÉKE .................................................................................................. 7 1. ÖSSZEFOGLALÁS ........................................................................................................... 9 1. SUMMARY .......................................................................................................................10 2. BEVEZETÉS ....................................................................................................................11 2.1. A téma jelentősége ......................................................................................................11 2.2. A kutatás célkitűzései .................................................................................................12 3. IRODALMI ÁTTEKINTÉS ................................................................................................13 3.1. A sertések vírusok okozta enterális megbetegedése ...............................................13 3.2. Enteropatogén kórokozók ..........................................................................................14 3.3. Immunszuppresszív vírusok .......................................................................................30 3.4. Enterális megbetegedéssel összefüggő egyéb vírusok ...........................................32 4. ANYAG ÉS MÓDSZER ....................................................................................................37 4.1. Mintagyűjtés ................................................................................................................37 4.2. Molekuláris biológiai módszerek ................................................................................40 4.3. Szerológiai módszerek ................................................................................................43 4.4. Vírusizolálás ................................................................................................................44 4.5. Statisztikai elemzések .................................................................................................45 5. EREDMÉNYEK ................................................................................................................46 3 5.1. Felmérő vizsgálat ........................................................................................................46 5.2. TGEV szerológia ..........................................................................................................52 5.3. PEDV esetekhez kapcsolódó eredmények ................................................................55 6. MEGBESZÉLÉS ..............................................................................................................60 6.1. A sertés enterális megbetegedésével összefüggő vírusok prevalenciája ...............60 6.2. A TGEV szerológiai felmérése ....................................................................................63 6.3. PEDV esetek ................................................................................................................64 7. ÚJ TUDOMÁNYOS EREDMÉNYEK ................................................................................69 8. HIVATKOZÁSOK JEGYZÉKE .........................................................................................70 9. A DOKTORI KUTATÁS EREDMÉNYEINEK KÖZLÉSEI .................................................97 10. MELLÉKLETEK .............................................................................................................99 11. KÖSZÖNETNYILVÁNÍTÁS .......................................................................................... 114 4 ÁBRÁK JEGYZÉKE 1. ábra: Súlyos hasmenés és kiszáradás jeleit mutató szopós malacok. ..............................13 2. ábra: Coronavírusok genomszerveződése. ......................................................................14 3. ábra: Coronavírusok elektronmikroszkópos felvétele. .....................................................15 4. ábra: Egészséges és kóros vékonybél szövettani képe. ..................................................17 5. ábra: TGEV és PRCV részleges genomjának az összehasonlítása. ................................17 6. ábra: Rotavírusok elektronmikroszkópos felvétele. ..........................................................25 7. ábra: Rotavírus szerkezete. .............................................................................................26 8. ábra: Astrovírus kapszid modellje. ...................................................................................32 9. ábra: Kobuvírus genomelrendeződése és sematikus szerkezete. ....................................35 10. ábra: Bélsárminták származási helye. ............................................................................37 11. ábra: Szérumminták származási helye. ..........................................................................38 12. ábra: PBoV3-4 kimutatására szolgáló PCR eredménye. ................................................46 13. ábra: PAstV kimutatására szolgáló PCR eredménye. ....................................................47 14. ábra: PEDV – FAM csatorna mérési eredménye. ...........................................................48 15. ábra: PDCoV – VIC/HEX csatorna mérési eredménye. ..................................................48 16. ábra: Belső RNS kontroll – Cy5 csatorna mérési eredménye. ........................................49 17. ábra: TGEV – VIC/HEX csatorna mérési eredménye. ....................................................49 18. ábra: A felmérő vizsgálat mintái telepi bontásban. .........................................................50 19. ábra: A felmérő vizsgálat mintái vírus és egészségügyi állapot szerint. ..........................51 20. ábra: A felmérő vizsgálat vírus- és mintaszám megoszlása korcsoport szerint. .............51 21. ábra: TGEV szerológia eredményének térképes megjelenítése. ....................................53 22. ábra: Anti-TGEV pozitív szérumminták száma telepenként. ...........................................53 23. ábra: A füzesabonyi sertéstelep szérummintáinak kvantitatív IFT eredménye. ...............54 24. ábra: Detektált rekombináció a PEDV HUN/5031/2016 S génjében. ..............................57 25. ábra: 26 reprezentatív PEDV teljes genom felhasználásával készült filogenetikai fa. .....59 5 TÁBLÁZATOK JEGYZÉKE 1. táblázat: A felmérő vizsgálat mintái kor és egészségügyi állapot szerint ..........................37 2. táblázat: A felmérő vizsgálathoz kiválasztott vírusok primerei. .........................................41 3. táblázat: A felmérő vizsgálat során alkalmazott PCR reakcióelegyek ..............................42 4. táblázat: A felmérő vizsgálat során alkalmazott PCR kondíciói ........................................42 5. táblázat: A felmérő vizsgálat PCR-pozitív mintái ..............................................................52 6. táblázat: Füzesabonyból származó szérumminták megoszlása. ......................................54 7. táblázat: A 2018-as PEDV esetekben érintett ’B’ és ’C’ farm vizsgálati eredményei ........56 6 RÖVIDÍTÉSEK JEGYZÉKE Jelentés Rövidítés Idegen nyelvű Magyar Acc. No. GenBank accession number génbanki azonosító szám Állategészségügyi Diagnosztikai ÁDI - Igazgatóság APN aminopeptidase N aminopeptidáz N BSA bovine serum albumin szarvasmarha szérum albumin Centers for Disease Control and CDC Amerikai Járványügyi Központ Prevention CPE cytopathogen effect citopatogén hatás Ct threshold cycle küszöb ciklus CV calicivirus calivivírus db - darab DMEM Dulbecco's Modified Eagle Medium szövettenyészet tápfolyadék DNS - dezoxi-ribonukleinsav E envelope burok ELISA enzyme-linked immunosorbent assay - FBS foetal bovine serum fötális szarvasmarha savó FITC fluorescein isothiocyanate fluoreszcein-izotiocianát HBoV human bocavirus humán bocavírus HE hemagglutinin esterase hemagglutinin-észteráz International Committee on Taxonomy Nemzetközi Vírusrendszertani ICTV of Viruses Bizottság IFN interferon interferon IFT immunofluorescence test immunfluoreszcencia teszt IgG immunoglobulin G immunglobulin G kb kilobase kilobázis km kilometer kilométer L leader (protein) vezető (fehérje) M membrane membrán min minute perc N nucleocapsid nukleokapszid Nemzeti Élelmiszerlánc-biztonsági NÉBIH - Hivatal NoV norovirus norovírus NP, NS, non-structural protein nem strukturális fehérje NSP nt nucleotide nukleotid OIE Office International des Epizooties Állategészségügyi Világszervezet ORF open reading frame nyitott olvasási keret PAdV porcine adenovirus sertés adenovírus PAstV porcine astrovirus sertés astrovírus PBoV porcine bocavirus sertés bocavírus PBS phosphate buffered saline foszfáttal pufferolt sóoldat PCR polymerase chain reaction polimeráz láncreakció 7 PCV porcine circovirus sertés circovírus sertés circovírushoz kapcsolódó PCVAD porcine circovirus associated
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