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VYTAUTO DIDŽIOJO UNIVERSITETAS Irma Pūraitė VYTAUTO DIDŽIOJO UNIVERSITETAS GAMTOS MOKSL Ų FAKULTETAS BIOLOGIJOS KATEDRA Irma P ūrait ė ELNINI Ų GENETIN Ė ĮVAIROV Ė BEI J Ų PERNEŠAMŲ ERKI Ų UŽSIKR ĖTIMAS BORRELIA BURGDORFERI S.L. IR ANAPLASMA PHAGOCYTOPHILUM Magistro baigiamasis darbas Molekulin ės biologijos ir biotechnologijos studij ų programa, 62401B107 Biologijos studij ų kryptis Vadovas prof. Algimantas Paulauskas (parašas) (data) Apginta prof. habil. dr. Gintautas Kamuntavi čius (parašas) (data) KAUNAS, 2010 Darbas atliktas: 2008 – 2010 m. Vytauto Didžiojo universitete, Biologijos katedroje, Kaune, Lietuvoje ir 2009.07.01 – 2009.09.30 LLP Erasmus student ų praktikos metu, Telemark University College, Department of Environmental and Health Studies, Bø i Telemark, Norvegijoje. Recenzentas: dr. J.Radzijevskaja, fiziniai mokslai, biochemija. Darbas ginamas: viešame magistr ų darb ų gynimo komisijos pos ėdyje 2010 met ų birželio 3d. Vytauto Didžiojo universitete, Biologijos katedroje, 801 auditorijoje, 10 val. Adresas: Vileikos g. 8, LT-44404 Kaunas, Lietuva. Protokolo Nr. Darbo vykdytojas: I.P ūrait ė Mokslinis vadovas: prof. A.Paulauskas Biologijos katedra, ved ėjas: prof. A.Paulauskas 2 TURINYS SANTRAUKA .................................................................................................................................. 4 SUMMARY ...................................................................................................................................... 5 ĮVADAS ............................................................................................................................................ 6 I. LITERAT ŪROS APŽVALGA ...................................................................................................... 8 1.1. Tauriojo elnio (Cervus elaphus) bendroji charakteristika ..................................................... 8 1.2. Stirnos (Capreolus capreolus) bendroji charakteristika ...................................................... 13 1.3. Iksodini ų erki ų (Ixodes ricinus) bendroji charakteristika .................................................... 19 1.3.1. Erki ų platinama Borrelia burgdorferi .......................................................................... 22 1.3.2. Erki ų platinama Anaplasma phagocytophilum ............................................................ 23 1.4. Elnini ų tyrimai ..................................................................................................................... 23 1.4.1 Elnini ų monitoringas Lietuvoje .................................................................................... 23 1.4.2. Elnini ų genetiniai tyrimai ............................................................................................. 25 1.4.3. Elnini ų užsikr ėtimas Ixodes ricinus erk ėmis ir j ų pernešamais patogenais ................. 30 II. MEDŽIAGA IR METODIKA .................................................................................................... 33 2.1. Tyrimo medžiaga ................................................................................................................. 33 2.2. Elnini ų DNR išskyrimas, koncentracijos ir švarumo nustatymas ........................................ 35 2.3. Erki ų ir j ų endoparazit ų DNR išskyrimas ............................................................................ 36 2.4. Elnini ų AAPD – PGR eiga .................................................................................................. 36 2.5. Borrelia burgdorferi s.l. PGR ............................................................................................... 38 2.5.1. Borrelia burgdorferi s.l. genotipavimas ...................................................................... 39 2.6. Anaplasma phagocytophilum PGR ...................................................................................... 40 2.7. Elektroforez ė agaroz ės gelyje. Fragment ų vizualizavimas .................................................. 41 2.8. Matematinis APPD rezultat ų įvertinimas ............................................................................ 42 III. REZULTATAI IR J Ų APTARIMAS ........................................................................................ 43 3.1. Tauri ųjų elni ų ir stirn ų genetin ė įvairov ė pagal AAPD analiz ę ........................................... 43 3.2. Erki ų užsikr ėtimas Borrelia burgdorferi s.l ........................................................................ 55 3.2.1. Borrelia burgdorfferi s.l. genotip ų identifikacija ......................................................... 56 3.3. Erki ų užsikr ėtimas Anaplasma phagocytophilum ................................................................ 57 IŠVADOS ........................................................................................................................................ 58 LITERAT ŪROS ŠALTINIAI ......................................................................................................... 59 PRIEDAI ......................................................................................................................................... 65 3 SANTRAUKA Darbo autorius: Irma P ūrait ė Pilnas darbo pavadinimas: Elninių genetin ė įvairov ė bei j ų pernešam ų erki ų užsikr ėtimas Borrelia burgdorferi s.l. ir Anaplasma phagocytophilum Darbo vadovas: prof. Algimantas Paulauskas Darbas ginamas: Vytauto Didžiojo Universitete, Gamtos moksl ų fakultete, Kaunas, 2010, birželio 3d. Puslapi ų skai čius: 64 Lenteli ų skai čius: 13 Paveiksl ų skai čius: 40 Pried ų skai čius: 10 Elninių genetinei įvairovei nustatyti buvo tiriami 27 tauri ųjų elni ų ir 36 stirn ų individai. Tauri ųjų elni ų ir stirn ų pavyzdžiai surinkti 2006-2009 metais Lietuvoje ir 2007 m. Norvegijoje. Individai tirti naudojant AADP (atsitiktinai amplifikuotos DNR polimorfizmo) ir PGR (polimeraz ės grandinin ė reakcija) metodik ą. Tyrimams naudoti kepen ų ir raumen ų pavyzdžiai. Stirn ų ir tauri ųjų elni ų individai buvo analizuojami naudojant dešimt pradmen ų: ROTH-180-01, ROTH-180-02, ROTH-180-03, ROTH-180-04, ROTH-180-05, ROTH-180-06, ROTH-180-07, ROTH-180-08, ROTH-180-09, ROTH-180-10. Elektroforez ės geli ų rezultatai parod ė, kad polimorfini ų lokus ų skai čius yra 79, o fragment ų dydis - nuo 150 iki 3000 bazi ų por ų. Nusta čius fragment ų dydžius ir skai čius, gauti duomenys buvo suvesti į „TREECON for Windows“ kompiuterin ę program ą, kuria remiantis įvertintas genetinis kintamumas. Genetinis kintamumas tarp tirtų tauri ųjų elni ų individ ų varijavo nuo 0,08 iki 0,80, tarp stirn ų – nuo 0,04 iki 0,76. Buvo įvertintas ir 75 Ixodes ricinus erki ų, surinkt ų nuo elnini ų, užsikr ėtimas Borrelia burgdorferi s.l. ir Anaplasma phacytophilum . 2,7% tirt ų erki ų buvo infekuotos Borrelia burgdorferi s.l., o 1,3% erki ų – Anaplasma phagocytophilum . 4 SUMMARY Author of term paper: Irma P ūrait ė Full title of term paper: Genetic diversity of deer and identification of Borrelia burgdorferi s.l. and Anaplasma phagocytophilum in ticks collected from deer Term paper adviser: prof. Algimantas Paulauskas Presented at: Vytautas Magnus University, Faculty of Naturas Sciences, Kaunas, 2010, June 3. Number of pages: 64 Number of tables: 13 Number of pictures: 40 Number of appendixes: 10 The Red deer ( Cervus elaphus ) 27 individuals from Lithuania and Norway and the Roe deer ( Capreolus capreolus ) 36 individuals from Lithuania were analyzed by RAPD (random amplified polymorphic DNA) polymerase chain reaction (PCR). The liver and muscle tissues samples were used for the investigations. Cervid individuals were analyzed using ten primers (ROTH-180-01, ROTH-180-02, ROTH-180-03, ROTH-180-04, ROTH-180-05, ROTH-180-06, ROTH-180-07, ROTH-180-08, ROTH-180-09, ROTH-180-10). Electrophoresis gels showed that the number of polymorphic loci are 79 and fragments size are from 150 to 3000. „TREECON for Windows“ programm was used for the estimation of genetic variability. Genetic variability among the Red deer separate individual varied from 0,08 to 0,80 and among the Roe deer varied from 0,04 to 0,76. A total 75 Ixodes ricinus ticks collected from cervids were examined for Borrelia burdorferi s.l. and Anaplasma phagocytophilum . Borrelia burgdorferi s.l. were detected in 2 ticks (2,7%) and Anaplasma phagocytophilum in 1 tick (1,3%). 5 Įvadas Lietuvos kanopini ų žv ėri ų fauna, susiformavusi po ledynme čio, iki ši ų dien ų smarkiai pakito. Tai įvyko d ėl gamtos ir klimato s ąlyg ų kitimo, v ėliau – dėl žmoni ų tiesiogin ės ir netiesiogin ės veiklos. Ištirpus paskutiniam ledynui, šalto klimato s ąlygomis, Lietuvos teritorijoje gyveno tundros ir miškatundr ės žv ėrys – avijau čiai, šiauriniai elniai, kurie v ėliau, klimatui atšilus, išnyko arba pasitrauk ė toliau į šiaur ę. XVI-XVII a. Lietuvoje išnyko stumbrai ir taurieji elniai (v ėliau šie žv ėrys mūsų krašte vėl buvo prad ėti auginti) (Baleišis ir kt., 1998). Šiandien Lietuvoje gyvenantiems elniniams (stirnoms ir tauriesiems elniams) pavojus išnykti negresia, j ų populiacijos gausios ir paplitusios beveik visuose Lietuvos miškuose. Ši ų žv ėri ų populiacijos yra puikus modelis genetinių padarini ų tyrimams. Lietuvos tauri ųjų elni ų gen ų fondas paveiktas reintrodukcijos, perk ėlimo, gyv ūnų laikymo aptvaruose, fragmentacijos bei medžiokl ės. Stirn ų populiacijos gen ų fondas paveiktas fragmentacijos ir medžiokl ės. Pastaraisiais dešimtme čiais Lietuvoje buvo tiriama kanopini ų biologija, morfologija bei ekologija ir j ų reikšm ė kult ūriniam kraštovaizdžiui (Baleišis ir kt., 2003). Elnini ų genetin ės įvairov ės tyrimai prad ėti vykdyti
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