Morfološke promene u tkivima barskih kornjača (Еmys orbicularis) prirodno inficiranih hemoparazitom Haemogregarina stepanowi i molekularno-genetička identifikacija uzročnika

Abstract

Hemoparaziti iz familije Haemogregarinidae često parazitiraju kod reptila, a u našoj zemlji do sada ne postoje podaci o infekciji uzročnicima hemoparazitom Haemogregarina stepanowi, kod jedine zaštićene vrste kornjača - evropske barske kornjače (Emys orbicularis), ni u prirodnim staništima, ni u kontrolisanim uslovima (zoo vrtovi, karantin). Klinički simptomi infekcije kornjača ovim hemoparazitom su nespecifični, nedostaju podaci o potencijalnim vektorima i o tome kakvu opasnost oni nose za druge životinje i čoveka. Ispitivanjima je obuhvaćena populacija od trideset barskih kornjača poreklom iz prirodnih staništa, a privremeno smeštenih u karantinu Zoo vrta Beograd, sa simptomima opšte slabosti i otežanog kretanja. Šest zdravih barskih kornjača, služile su kao kontrolna grupa za kliničke i citološke analize kao i molekularno-genetička ispitivanja. Životinje su pažljivo makroskopski pregledane i markirane radi kasnije identifikacije. Nakon uzorkovanja krvi vraćene su na mesto gde su izlovljene u skladu sa čl.78. Zakona o zaštiti prirode (“Službeni glasnik RS”, br.5/10, 47/11). Za ovu vrstu ispitivanja je dobijena dozvola – rešenje br. 119- 01-13-2-2015-09 od 12.01.2015. od Ministarstva poljoprivrede i zaštite životne sredine. Molekularno-genetičkim analizama (PCR) ustanovljeno je prisustvo Haemogregarina stepanowi u uzorcima krvi svih trideset odraslih kornjača oba pola. Molekularno-genetička ispitivanja uzoraka krvi urađena su u cilju identifikacije hemoparazita Haemogregarina stepanowi. Izolacija DNK iz parafinskih isečaka tkiva 5 μm i krvi ispitivanih kornjača rađena je primenom komercijalnog seta “KAPA Express Extract Kit” prema protokolu proizvođača. Ispitivanje prisustva nukleinske kiseline parazita Haemogregarina stepanowi urađeno je kod ukupno 36 uzoraka krvi barskih kornjača (Emys orbicularis). Primenom prajmera EF: (5’ – GAAACTGC- GAATGGCTCATT -3) i ER: (5’- CTTGCGCCTACTAGGCATTC-3’) amplifikovani su 1500 bp dugački fragmenti jedarne 18S rDNK. Nakon vizualizacije na 1,5% agaroznom gelu, kod 30 ispitivanih uzoraka amplifikati su bili prisutni. Nakon amplifikacije željenog segmenta DNK parazita, dobijeni amplifikati su iskorišćeni za sekvencioniranje željnog segmenta gena odgovornog za sintezu 18S ribozomalne DNK. Rezultati sekvencioniranja (Gen Bank Number KT749877) potvrdili su da su svi ispitivani uzorci 100% identični sekvencama 18S ribozomalne DNK, koje su prethodno prijavljene u genskoj bazi od strane drugih autora (KF257929. 1, KF257927.1)...

Hemoparasites from the Haemogregarinidae family often parasite on reptiles, in our country there is no data on infection of the only protected turtle – European pond turtle (Emys orbicularis) caused by Haemogregarina stepanowi, neither in their natural habitat nor under controlled conditions (zoo gardens, quarantine). Clinical symptoms of the infection caused by this hemoparasite are not specific and there is a lack of information on potential vectors and on the risk they pose to other animals and humans. The investigations covered a population of thirty pond turtles originating from their natural habitat, temporarily placed in quarantine at the Belgrade Zoo, with symptoms of general weakness and difficulty in movement. Six healthy pond turtle, served as a control group for clinical and cytological analysis and molecular genetic testing. The animals were carefully examined macroscopically and marked for later identification. Six healthy pond turtles served as the control group for clinical and cytological analysis and molecular genetic testing. After blood sampling they were returned to the place where they were caught, in accordance to article 78, Nature Protection Act (“Official Gazette”, No.5 / 10, 47/11). For this type of testing in a permission has been obtained - No. 119-01-13-2-2015-09 dated 12.01.2015 by the Ministry of Agriculture and Environmental Protection. Molecular genetic analysis (PCR) established the presence of Haemogregarina Stepanowi in the blood samples of all thirty adult turtles of both sexes. Molecular - genetic tests of blood samples were carried out in order to identify Haemogregarina stepanowi. Isolation of DNA from tissue samples embedded in paraffin and measuring 5 microns blood samples tests was done using the commercial set “KAPA Express Extract kit” according to the manufacturer’s protocol. Detection of Haemogregarina Stepanowi nucleic acids was done on a total of 36 blood samples of the pond turtle (Emys orbicularis). By applying the primer EF: (5 ‘- GALACTIC- GAATGGCTCATT -3) and ER (5 ‘- CTTGCGCCTACTAGGCATTC-3’) amplified 1500 bp long fragments of the nuclear 18S rDNA. After visualization on a 1.5% agarose gel in the 30 tested samples amplified molecules were present. After amplification of the desired parasite DNA segments, the obtained amplified molecules were used for sequencing the desired segment of the gene responsible for the synthesis of 18S ribosomal DNA...