Molekularna tipizacija piroplazmi iz postmortalnih citoloških otisaka organa i razmazaka periferne krvi pasa bojanih po giemsi

master's thesis

• Title (english): Molecular Typing for Piroplasms Detected in Giemssa Stained Postmortem Obtained Tissue Inprints and Peripheral Blood Smears Originateing from Dogs
• Author: Željka Anzulović
• Mentor: Ana Beck (mentor)
• Mentor: Relja Beck (mentor)
• Committee member: Andrea Gudan Kurilj
• Committee member: Relja Beck
• Committee member: Ana Beck
• Granter: University of Zagreb; Faculty of Veterinary Medicine; (Department of Veterinary Pathology); Zagreb
• Defense date and country: 2014-11-14, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Basic and Preclinical Veterinary Sciences

Abstract

Babezioza je najčešći uzročnik hemolitičkih anemija u Hrvatskoj. Temeljem morfologije merozoita babezije/teilerije dijele se na velike i male piroplazme. Za razliku od razmazaka periferne krvi u otiscima organa nije moguće razlikovati velike od malih babezija, jer mijenjaju svoja morfološka obilježja. Morfološki nije moguće razlikovati vrste odnosno podvrste , stoga je nužno koristiti molekularne metode za gensku tipizaciju uzročnika hemolitičke anemije u pasa.Ovo se najčešće provodi iz svježih uzoraka krvi no kojiput je potrebno prevesti gensku tipizaciju iz razmazaka krvi odnosno otisaka organa i tkiva uginulih pasa. U ovom istraživanju provedena je genska tipizacija iz 22 strugotine arhivskih citoloških uzoraka s tri različita protokola. U svim uzorcima prethodno je mikroskopski analizirana staničnost uzorka, prisutnost merozoita te njihova morfologija. Od 13 citološki pozitivnih uzoraka DNK je različitim protokolima umnožena u 12 uzoraka. Genska tipizacija je provedena na 9 uzoraka uspješno umnoženih trećim protokolom. DNK babezija je umnožena pomoću tri različita protokola: (I) jednostruka PCR reakcija (II) dvostruka PCR reakcija „nested“ (III) jednostruka PCR reakcija iz pročišćenih i koncentriranih DNK uzoraka. Uspješnost umnažanja je bila različita i iznosila je 31%, 70% i 70%. Također su dokazane razlike u uspješnosti umnažanja između otisaka organa i krvnih razmazaka istim protokolom. Tako je drugim protokolom iz krvnih razmazaka uspješnost umnažanja iznosila 60%, a iz otisaka organa 75%. Trećim protokolom iz krvnih razmazaka je uspješnost bila 40%, a iz otisaka 88%. Različita uspješnost umnažanja ukazuje na nužnost primjene različitih molekularnih protokola za dokazivanje piroplazmi u otiscima organa i razmascima krvi. Uspješnost umnažanja je potvrđena analizom nukleotidnih sljedova i dokazivanjem protozona B. canis canis. Ovim istraživanjem dokazana je prikladnost genske tipizacije i analize nukletidnih sljedova patogena. Analiza nukleotidnih sljedova prikladna je za dokazivanje vrste/ podvrste patogena iz arhivskih citoloških uzorcaka obojenih po Giemsi ili Diff Quick®.

Abstract (english)

Babesiosis is common infectious agents of haemolytic anaemia in dogs in Croatia. Based on morphology of merozoites, Babesia/Theilera species are divided on big and small piroplasms. Differentiation between big of small piroplasms is not possible in the tissue imprints, unlike in peripheral blood smear, because they change their morphologic characteristic. The use of molecular methods for genetic typing of causative agent of haemolytic anaemia in dogs is necessary because morphologically is not possible to distinguish species, or subspecies of piroplasms. This is generally done from the fresh blood samples, but sometimes is necessary to perform genetic typing from the blood smear, or post-mortem tissue imprints. In this study genetic typing is performed from 22 grinds of cytological samples using three different protocols. Previously, cellularity, merozoites finding and their morphology have been analysed in all samples by microscopic method. Using different PCR protocols DNA is duplicated in 12 samples from 13 cytological positive samples. Genetic typing is performed on 9 samples successfully duplicated in third protocol. DNA of Babesiosis is duplicated using three different protocols: (I) simple PCR reaction (II) double PCR reaction „nested“ (III) simple PCR reaction from purified and concentrated DNA samples. The successfulness of DNA multiplication was different and amounted 31%, 70% and 70%. Significant differences are established in successfulness of multiplication between tissue imprints and blood smear using the same protocol. Using the second protocol successfulness of multiplication in blood smear samples was 60%, and from tissue imprints 75%. In third protocol successfulness of multiplication in blood smears samples was 40% and in tissue imprints samples 88%. The difference in the successfulness of duplication points on necessity of using different molecular protocols for detection of piroplasms in the tissue imprints and blood smear. The successfulness is confirmed with analysis of nucleotide sequences and detection of protozoon B. canis canis. In this study suitability of genetic typing and analysis of nucleotide sequences of pathogens is detected. The analysis of nucleotide sequences is suitable for detection of species/ subspecies of piroplasms from archival cytological samples stained by Giemsa or Diff Quick®.

• Keywords: Piroplazmoza; Babesia canis canis; arhivski citološki uzorci; genska tipizacija
• Keywords (english): Piroplasmosis; Babesia canis canis; archival cytological samples; PCR
• Language: croatian
• URN:NBN: urn:nbn:hr:178:992638
• Study programme: Title: Veterinary Medicine; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: doktor/doktorica veterinarske medicine (doktor/doktorica veterinarske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2015-12-02 13:15:59