Molekularna dijagnostika i filogenetska tipizacija virusa papilomatoze pasa

master's thesis

• Title (english): Detection, Identification and Molecular Typing of Canine Papillomavirus in Croatia
• Author: Marina Dabić
• Mentor: Ljubo Barbić (mentor)
• Committee member: Zoran Milas
• Committee member: Vladimir Stevanović
• Committee member: Ljubo Barbić
• Granter: University of Zagreb; Faculty of Veterinary Medicine; (Department of Microbiology and Infectious Diseases with Clinic); Zagreb
• Defense date and country: 2015-12-04, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Veterinary Medicine; Veterinary Clinical Sciences

Abstract

Papilomavirusi pasa uzrokuju niz razlicitih klinickih promjena opisanih u svijetu, no najcešce
prepoznato je stvaranje bradavica u usnoj šupljini i na koži. U posljednje vrijeme cesta je pojava
neoplazmi nazvanih epidermalni pigmentirani plakovi koji su potaknuti papilomavirusom. Do danas je
u svijetu izolirano 16 razlicitih virusnih tipova. Premda klinicki izrazito specificne i lako
prepoznatljive do danas u Republici Hrvatskoj nikada nije napravljeno istraživanje o ucestalost virusne
etiologije u nastanku bradavica.
Istraživanje o zastupljenosti pojedinog tipa pseceg papilomavirusa napravljeno je pretraživanjem
uzoraka tkiva 22 papiloma pasa koji su kirurški uklonjenih tijekom klinicke obrade životinja. Svi
uzorci su dostavljeni u virusološki laboratorij Zavoda za mikrobiologiju i zarazne bolesti sa klinikom
Veterinarskog fakulteta gdje se provela daljnja obrada sa svrhom izrade autovakcine. Prema
lokalizaciji promjena pet pasa je imalo bradavicu u usnoj šupljini, jedan na gornjoj vjedi, a ostatak
bradavica uklonjeno je s kože životinja. Pošto se papilomavirusi ne mogu izolirati na stanicnim
kulturama, za etiološku dijagnozu korištena je lancana reakcija polimerazom u svrhu dokazivanja
virusne DNK.
Za izdvajanje virusnog DNK korišten je komercijalni komplet QIAamp DNA Mini Kit prema uputama
proizvodaca. Molekularna dijagnostika CPV nacinjena je PCR metodom korištenjem u ranijim
istraživanjima opisanih pocetnica FAP59 i FAP64. Pocetnice su specificne za L1 gen ORF regije
virusa papilomatoze. Dobiveni PCR produkti poslani su na sekvencioniranje, a odredeni nukleotidni
slijedovi usporedeni su s nukleotidnim slijedovima do sada tipiziranih CPV dostupnim u GeneBank
radi filogenetske analize.
U 18 od 22 uzoraka je dobivena pozitivna reakcija, dok je cetiri uzorka pokazalo nespecificnu reakciju.
Virusna etiologija dokazana je u 81.9% pretraženih uzoraka. U uzorcima papiloma iz usne šupljine i
jednog s vjede je dokazan virus, dok se u kožnim promjenama virus dokazao u 12 od 16 (75%). Svi
tipizirani CPV filogenetskom analizom pripadaju CPV tip 1, unutar skupine Lambdapapillomavirus.
Uocena je minimalna medusobna razlikama na nukleotidnoj, ali i aminokiselinskoj razini.
59
Istraživanjem je dokazano da je vecina papilomatoznih promjena u pasa u RH uzrokovana CPV tip 1.
Promjene bez potvrdene virusne etiologije dokazane su na koži. Dokazivanjem pripadnosti svih
pretraženih uzoraka, CPV 1 tipu virusa, neovisno o lokalizaciji izazvane promjene kao ni geografskog
podrucja zaprimanja uzoraka, postavlja se temelj za izradu autovakcine bazirane na CPV1 tipu virusa,radi zaštite neimunih životinja kao i ubrzavanja procesa oporavka vec oboljelih.

Abstract (english)

Canine papilomavirus is common cause of wart formation in oral cavity and skin of dogs. By now, 16
different virus types have been isolated. Although clinically highly specific and easily recognized, an
incidence of viral papilomas in dog population in Croatia has been never determined.
A total of 22 papiloma samples sent to Virology laboratory, Faculty of Veterinary Medicine, Zagreb,
for the autovaccine production were tested. Samples were surgically removed at local veterinary
clinics: five from oral cavity, one from the upper eyelid, and the rest were removed from the skin.
Polymerase chain reaction (PCR) was used to prove viral DNA, and thus confirm the viral aetiology.
Sample DNA was isolated using commercial kit QIAamp DNA Minit Kit, according to manufacturers'
instructions. For the specific amplification of L1 gene ORF region of the papillomavirus earlier
described primers FAP59 and FAP64 were used. The obtained PCR products were sequenced and
compared with corresponding nucleotide sequences available in the GeneBank for the phylogenetic
analysis.
Viral aetiology was proven in 18 out of 22 samples (81.9%) of analysed samples. The virus was
identified in all of the samples obtained from oral cavities, the sample taken from the upper eyelid and
in 12 out of 16 (75%) skin lesions.
Sequences showed substantial difference at both nucleotide and amino-acid level even though
amplified region is highly conserved one.
All of the CPV isolates from this study belong to CPV type 1, in the group of Lambdapapillomavirus.
Even based on small number of samples its clear most of papillomatous lesions in dogs in Croatia have
viral aetiology. Interestingly all the lesions without confirmed viral aetiology have originated from a
skin. Results of this research can be used as a guidance in a wart treatment and considerable variability
of isolated CPV highlight advantage of autovaccine usage if immunotherapy is considered.

• Keywords: papilomatoza pasa; lancana reakcija polimerazom; molekularna tipizacija; Hrvatska
• Keywords (english): canine papillomatosis; PCR; molecular typisation; Croatia
• Language: croatian
• URN:NBN: urn:nbn:hr:178:822766
• 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: 2016-01-12 08:54:41