ࡱ> BD=>?@Avbjbj|| 4F"+     !!!8Y\!>09Oeee@Z f=======@BF= n@@nn=  eeq>8<<<n e e=<n=<<<e!4<=><><B7hB<<B <nn<nnnnn==8:Vnnn>nnnnBnnnnnnnnn :GENOTIPOVI COXIELLA BURNETII U REPUBLICI HRVATSKOJ Ivana Ra i, Sanja Duvnjak, Silvio `pi i, Maja Zdelar-Tuk, Irena Reil, }eljko Cvetni Hrvatski veterinarski institut Laboratorij za bakterijske zoonoze i molekulsku dijagnostiku bakterijskih bolesti Bakterijska vrsta Coxiella (C.) burnetii je vrlo infektivna bakterija i obvezatni unutarstani ni parazit. U ljudi i ~ivotinja uzrokuje Q-groznicu koja je raairena po cijelom svijetu. U ovom radu su po prvi put genotipizirani sojevi vrste C. burnetii u Republici Hrvatskoj (RH) metodom MLVA (engl. MultiLocusVNTRAnalysis, VariableNumberofTandemRepeats) koja se u svijetu tek uvodi kao metoda za genotipizaciju C. burnetii. Metoda je primijenjena na 49 uzoraka podrijetlom od domaih ~ivotinja, 32 uzoraka je u potpunosti tipizirano, a njih 31 svrstano je u jedan od 13 dobivenih genotipova. Dva genotipa srodnija su s genotipovima u svijetu nego s hrvatskim genotipovima, a karakteristi ni su za kontinentalni dio Hrvatske. Ostali genotipovi srodniji su meusobno nego sa stranim genotipovima, a karakteristi ni su za obalnu Hrvatsku. Metoda MLVA uvedena je kao rutinski dijagnosti ki postupak ime je omogueno utvrivanje izvora i puteva airenja infekcije ato e pridonijeti kontroli i suzbijanju bolesti u domaih ~ivotinja koje su ujedno primarni izvor zaraze za ljude. UVOD Coxiella burnetii je iznimno infektivna bakterija koja u ljudi i ~ivotinja izaziva Q-groznicu. Ljudi i ~ivotinje se naj eae zaraze udisanjem aerosola i praaine zara~ene bakterijom. C. burnetii izlu uje se u mlijeku, vaginalnom iscjetku, fecesu, a pogotovo u posteljici zara~ene ~ivotinje te samom fetusu (Maurin i Raoult, 1999; Arricau-Bouvery i Rodolakis, 2005). Posteljica zara~enih ~ivotinja mo~e sad~avati i do 109 bakterija po gramu (Arricau-Bouvery i Rodolakis, 2005.). U Republici Hrvatskoj (RH) godianje je prijavljeno 20-70 slu ajeva Q-groznice u razdoblju od 2000.-2012., s vrhuncem od 206 slu ajeva u 2003. te 104 slu aja u 2004. godini. (Hrvatski zavod za javno zdravstvo, 2012). Ovim istra~ivanjem smo identificirali u Hrvatskoj prisutne genotipove sojeva C. burnetii izdvojenih iz terenskih uzoraka ljudi i domaih ~ivotinja te usporedili iste s dostupnim genotipovima iz raznih dijelova svijeta. Informacije dobivene ovim istra~ivanjem mogle bi u budunosti predvidjeti pojavu zaraza te pojasniti kako se Hrvatski sojevi odnose prema stranim sojevima. Takoer, nadamo se na taj na in doprinijeti i razvoju MLVA tehnike kao standardne metode genotipizacije ove bakterije. MATERIJALI I METODE U razdoblju od 2004. - 2012. izdvojili smo DNA iz 3261 uzorka razli itih tkiva ljudi i ~ivotinja koristei DNeasy Blood and Tissue (Qiagen) i QIAamp DNA Mini Kit (Qiagen). Svi uzorci provjereni su na prisutnost DNA C. burnetii konvencionalnim PCR-om (Willems i sur., 1994.) (Tablica 1.). Tablica 1. Podrijetlo i broj uzoraka tkiva provjeravanih na prisutnost DNA C. burnetii DomainBroj uzorakaProvjerenoPozitivno (%)*ljudi1926 (3.1)goveda160444 (2.7)koze739218 (29.5)ovce68148 (7.1)konji4519 (42.2)Ukupno3261335 (10.3)*Konvencionalnim PCR-om (Willems i sur., 1994.). Uzorci DNA koji su bili pozitivni, 82 je genotipizirano koriatenjem MLVA. Tehnika obuhvaa 17 razli itih lokusa opisanih od strane Arricau-Bouvery i sur. (2006.). Veli ina fragmenata DNA odreena je kapilarnom elektroforezom pomou ureaja QIAxcel ili klasi nom gel-elektroforezom.(Qiagen). Na osnovu veli ne DNA fragmenata odreen je broj ponavljanja za svaki lokus. Rezultate tipiziranja, u obliku 17- lanog koda, analizirali smo UPGMA algoritmom u Bionumerics 7.1 software-u (Applied Maths, Belgium). Izra unali smo i Hunter-Gaston indeks razmolikosti (HGDI) radi procjenjivanja diskriminacijskih mogunosti metode MLVA (Hunter i Gaston, 1988). REZULTATI Izolirali smo DNA iz 3261 uzorka tkiva ljudi i ~ivotinja te smo u njih 335 (10.3%) uspjeli identificirati DNA C. burnetii pomou konvencionalnog PCR-a (Willems i sur., 1994.). Od tih 335 uzoraka, 82 smo izabrali za genotipizaciju. Od izabranih uzoraka, samo 32 uzorka smo uspjeli genotipizirati na najmanje 16 lokusa. Od toga, 26 uzoraka smo uspjeli tipizirati na svih 17 lokusa. `est uzoraka nismo uspjeli genotipizirati na lokusu ms23. Odredili smo 13 razli itih genotipova. Distribucija inih u Hrvatskoj prikazana je na Slici 1. Procijenili smo mogunost diskriminacije svih lokusa u eksperimantalnim uvjetima ra unajui HGDI kako su opisali Arricau-Bouvery i sur. (2006.).  Slika 1. 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Identificirali smo 13 novih genotipova (CbCRO01  CbCRO13), sugerirajui da je globalna raznolikost C. burnetii vrlo podcijenjena. Genotip CbCRO01 izdvojen je iz najstarijeg uzorka u o~ujku 2004. Od svih 13 identificiranih genotipova, CbCRO01 je nasli niji CbCRO02. Ova dva genotipa se od ostalih razlikuju u 10 od 11 lokusa, ukazujui na nesrodnost s ostalim hrvatskim genotipovima. Nalaze se samo u kontinentalnom podru ju RH, posebice u Osje ko-baranjskoj, Koprivni ko-kri~eva koj, Vukovarsko-srijemskoj i Bjelovarsko-biologorskoj ~upaniji. Genotipovi CbCRO04, CbCRO08, CbCRO09, CbCRO10 and CbCRO12 razlikuju se samo na lokusu ms26. Svih pet genotipova pojavljuje se isklju ivo u priobalnom dijelu RH, osim CbCRO12, koji se pojavljuje i u kontinentalnom dijelu (Karlova ka ~upanija). Postoji mogunost da jedan genotip, CbCRO09, prelazi s ovaca na goveda. Identificirali smo ga 2000. na farmi koza u Splitko-dalmatinskoj ~upaniji te opet 2013. na farmi goveda u Bjelovarsko-biologorskoj ~upaniji. Pretpostavljamo da su ~ivotinje nezakonito prevezene s jedne farme na drugu te da je uzro nik preaao s koza na goveda. Genotipovi CbCRO04, CbCRO08, CbCRO09, CbCRO10 i CbCRO12 razlikuju se na 1 ili 2 lokusa od genotipova CbCRO07, CbCRO03, CbCRO05 i CbCRO11 dok se od CbCRO06 i CbCRO13 razlikuju na tri lokusa. Tih 11 genotipova pojavljuju se u priobalnom dijelu RH, osim CbCRO05, koji je prisutan u kontinentalnom podru ju (Zagreba ka ~upanija). U `ibensko-kninskoj ~upaniji identificirali smo najviae razli itih C. burnetii genotipova: CbCRO03, CbCRO04, CbCRO09, CbCRO10 i CbCRO11, dok smo u drugim ~upanijama identificirali najviae tri razli ita genotipa. Vrijednosti indeksa raznolikosti (HGDI) izra unali smo za svih 17 lokusa te je vrijednost vrlo visoka: 0.923. Usporeujui hrvatske genotipove s dostupnim genotipovima iz svih krajeva svijeta primijetili smo unikatnost hrvatskih sojeva. Genotipovi CbCRO02 i CbCRO01 najsrodniji su stranim genotipovima. Oni nisu srodni niti jednom drugom hrvatskom genotipu te se zadr~avaju isklju ivo u kontinentalnom dijelu Hrvatske. Svi drugi identificirani genotipovi srodniji su meusobno nego sa stranim genotipovima. Pretpostavljamo da su tih 11 genotipova endemi ni za Hrvatsku. Daljnja istra~ivanja su nu~na kako bi utvrdili moguu prisutnost hrvatskih genotipova u drugim zemljama za koje sada ne raspola~emo s podacima. Svakako bi trebalo doraditi trenuta no koriatenu MLVA shemu za tipizaciju Coxiella burnetii kako bi se poveala robustnost iste za razli ite vrste i kvalitetu uzoraka. References Arricau-Bouvery, N., Hauck, Y., Bejaoui, A., Frangoulidis, D., Bodier, C.C., Souriau, A., Meyer, H., Neubauer, H., Rodolakis, A., Vergnaud, G., 2006. Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing. BMC Microbiol 6, 38. Arricau-Bouvery, N., Rodolakis, A., 2005. Is Q fever an emerging or re-emerging zoonosis? Vet Res 36, 327-349. Croatian National Institute of Public Health, 2012. Croatian Health Service Yearbook 2012. p. 188.  HYPERLINK "http://hzjz.hr/wp-content/uploads/2013/11/ljetopis-2012.pdf" http://hzjz.hr/wp-content/uploads/2013/11/ljetopis-2012.pdf (accessed on March, 5th, 2014) Hunter, P.R., Gaston, M.A., 1988. Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26, 2465-2466. Maurin, M., Raoult, D., 1999. Q fever. Clin Microbiol Rev 12, 518-553. MLVA bank for Microbes Genotyping on the internet page http://mlva.u-psud.fr/mlvav4/genotyping/index.php (accessed on February, 2nd 2014) Ra i, I., `pi i, S., Galov, A., Duvnjak, S., Zdelar-Tuk, M., Vujnovi, A., Habrun, B., Cvetni, }., 2014. Identification of Coxiella burnetii genotypes in Croatia using multi-locus VNTR analysis. Vet Microbiol 173, 340 347. Willems, H., Thiele, D., Frlich-Ritter, R., Krauss, H., 1994. 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