Primena molekularnih metoda u dijagnostici bolesti kvrgave kože goveda na teritoriji Republike Srbije i njihov značaj u proceni epizootiološke situacije

Abstract

Bolest kvrgave kože (BKK) ili nodularni dermatitis (engl. Lumpy skin disease - LSD) jeekonomski važna virusna bolest goveda koja je juna meseca 2016. godine prvi put registrovanana teritoriji R. Srbije. Cilj ove doktorske disertacije bila je izolacija i identifikacija virusa BKK,sekvenciranje dela genoma izolovanog i identifikovanih virusa BKK i molekularnoepizootiološka analiza dobijenih podataka, kao i priprema i validacija novih protokola zaizvođenje real-time PCR metode za dokazivanje terenskog i vakcinalnog soja virusa i definisanjenajspecifičnijeg i najosetljivijeg protokola za dijagnostiku BKK. U okviru doktorske disertacijeurađena je epizootiološka analiza epizootije BKK na epizootiološkom području Veterinarskogspecijalističkog instituta Niš.Uzorci za ispitivanje (krv, bioptati kože i nosni brisevi) sakupljeni su od 148 goveda tokomepizootije BKK na epizootiološkom području VSI Niš. Ukupno je ispitano je 167 uzoraka od 82životinje sa kliničkim simptomima i 66 uzoraka krvi od 66 goveda bez kliničkih simptoma kojisu uzorkovani pre vakcinacije, kao i 40 uzoraka poreklom od 20 goveda kod kojih su se kliničkisimptomi pojavili nakon vakcinacije.Iz uzorka bioptata kože goveda sa ispoljenim kliničkim simptomima urađena je izolacija virusabolesti kvrgave kože. Izolovani virus (Pčinj 1) je identifikovan primenom klasičnih virusoloških(VNT) i molekularnih metoda (real-time PCR). Sekvenciranje delova genoma virusa BKK i točetiri gena: RPO30, GPCR, EEV i P32 urađeno je iz izolovanog virusa i šest uzoraka bioptatakože (pozitivnih real-time PCR) metodom po Sanger-u. Filogenetska analiza na osnovudobijenih sekvenci RPO30, GPCR, EEV i P32 gena pokazala je potpunu podudarnost uredosledu nukleotida izolovanog i šest identifikovanih virusa BKK, kao i potpunu podudarnostsa analognim sekvencama soja Serbia/Bujanovac/2016. Utvrđeno je da oba soja virusa izolovanana području R. Srbije (Serbia/Bujanovac/2016 i Pčinj 1), kao i virusi detektovani u bioptatimakože pripadaju podgrupi terenskih sojeva virusa BKK. Analizom filogenetskog stablaformiranog na osnovu poređenja celog genoma soja Serbia/Bujanovac/2016 sa celim genomima19 CaPV koji se nalaze u banci gena (GenBank) utvrđeno je da se soj Serbia/Bujanovac/2016nalazi u podgrupi sa terenskim sojevima virusa BKK, pri čemu je najveća podudarnost uredosledu nukleotida utvrđena sa sojevima izolovanim u Grčkoj, Izraelu, Ruskoj Federaciji iJužnoj Africi.Dizajniranje sekvenci prajmera i probe za novi real-time PCR protokol za detekciju terenskogsoja virusa (Terenski Niš) zasnovano je na razlikama u redosledu nukleotida na LSD008 genuizmeđu terenskih i vakcinalnih sojeva, dok je dizajniranje sekvenci prajmera i probe za novi realtimePCR protokol za detekciju vakcinalnog soja virusa (Vakcinalni Niš) zasnovano narazlikama u redosledu nukleotida na genu LSD146 terenskog i vakcinalnog soja virusa BKK.Ispitivanjem analitičke specifičnosti utvrđeno je da se protokolom Terenski Niš pored virusaBKK detektuju i virusi boginja i ovaca, ali se jasno diferenciraju terenski i vakcinalni soj virusaBKK. Real-time PCR protokol Vakcinalni Niš je specifičan samo za vakcinalne (Neethling)sojeve virusa BKK. Ispitivanjem analitičkih i dijagnostičkih performansi potvrđeno je da realtimePCR protokoli Terenski Niš i Vakcinalni Niš poseduju zadovoljavajuće performanse i moguse koristiti u dijagnostici BKK.Ispitivanje prisustva genoma virusa BKK iz uzoraka izvršeno je primenom šest protokola zaizvođenje real-time PCR metode (Bowden i sar., 2008, KV-2, Terenski Niš, KV-vac, VakcinalniNiš i komercijalnim real-time PCR) i protokolom za izvođenje nested PCR metode (Menasherowi sar., 2014)...

Registered in the territory of the Republic of Serbia in June 2016. The aim of this doctoraldissertation was the isolation and identification of LSD virus, sequencing part of the genome ofthe isolated and identified LSD virus and molecular epizootiological analysis of the obtaineddata, as well as preparation and validation of new protocols for real-time PCR method to detectfield and vaccine strain and define the most specific and sensitive protocol. An epizootiologicalanalysis of the LSD epizootic that occurred in the epizootiological area controlled by theVeterinary Specialized Institute Nis in 2016, was also conducted within this doctoraldissertation.Samples for testing (blood, skin biopsies and nasal swabs from LSD suspected or susceptiblecattle) were collected from 148 animals during the LSD epizootic in the epizootiological area ofVSI Nis. A total of 167 samples from 82 animals with clinical symptoms and 66 blood samplesfrom 66 animals without clinical symptoms that were sampled before vaccination, as well as 40samples from 20 animals in which clinical symptoms appeared after vaccination, were examined.Isolation of the Lumpy Skin Disease Virus was performed from a bovine skin biopsies thatoriginated from animals with clinical symptoms. The isolated virus (Pčinj 1) was identified usingclassical virological (VNT) and molecular diagnostic methods (real-time PCR). Sequencing ofparts of the LSDV genome, namely four genes: RPO30, GPCR, EEV and P32, was performedfrom the isolated virus and six skin biopsy samples (positive real-time PCR) by the Sangermethod. Phylogenetic analysis based on the obtained sequences of RPO30, GPCR, EEV and P32genes showed complete match in the sequence of nucleotides from isolated and six identifiedLSD viruses, as well as with analogous sequences of the Serbia/Bujanovac/2016 LSDV strain. Itwas determined that both strains of viruses isolated in the Republic of Serbia(Serbia/Bujanovac/2016 and Pčinj 1), as well as viruses detected in skin biopsies belong to thesubgroup of field strains of LSDV. Analysis of the phylogenetic tree formed on the basis ofcomparison of the whole genome of the strain Serbia/Bujanovac/2016 with the whole genomesof 19 CaPV located in the gene bank (GenBank) showed that the Serbia/Bujanovac/2016 strain isin a subgroup along with field strains of LSDV and has the highest nucleotide sequence matchwith LSDV strains isolated in Greece, Israel, the Russian Federation and South Africa.The design of primer and probe sequences for the new real-time PCR protocol for field virusstrain detection (Terenski Niš) was based on differences in the nucleotide sequence of theLSD008 gene between field and vaccine strains, while the design of primer and probe sequencesfor the new vaccine strain detection protocol (Vakcinalni Nis) was based on differences in thesequence of nucleotides on the LSD146 gene from both field and vaccine LSDV strains. Byexamining the analytical specificity, it was determined that the Terenski Niš protocol detectsGoatpox and Sheeppox viruses in addition to the LSDV, but clearly differentiates the field andvaccine strain of the LSDV. Real-time PCR protocol Vakcinalni Nis is specific only for vaccine(Neethling) strains of LSDV. Analytical and diagnostic performance testing confirmed that thereal-time PCR protocols Terenski Niš and Vakcinalni Nis have satisfactory performance and canbe used in the diagnosis of LSD.Examination for the presence of LSDV genomes from the samples was performed using sixdifferent real-time PCR protocols (Bowden et al., 2008, KV-2, Terenski Niš, KV-vac,Vakcinalni Niš and commercial real-time PCR) as well as one nested PCR protocol(Menasherow et al., 2014)...