The application of molecular diagnostic methods in the diagnosis of lumpy skin disease in the territory of the Republic of Serbia and their significance for the assessment of epizootiological situation

Bolest kvrgave kože (BKK) ili nodularni dermatitis (engl. Lumpy skin disease - LSD) je ekonomski važna virusna bolest goveda koja je juna meseca 2016. godine prvi put registrovana na teritoriji R. Srbije. Cilj ove doktorske disertacije bila je izolacija i identifikacija virusa BKK, sekvenciranje dela genoma izolovanog i identifikovanih virusa BKK i molekularno epizootiološka analiza dobijenih podataka, kao i priprema i validacija novih protokola za izvođenje real-time PCR metode za dokazivanje terenskog i vakcinalnog soja virusa i definisanje najspecifičnijeg i najosetljivijeg protokola za dijagnostiku BKK. U okviru doktorske disertacije urađena je epizootiološka analiza epizootije BKK na epizootiološkom području Veterinarskog specijalističkog instituta Niš. Uzorci za ispitivanje (krv, bioptati kože i nosni brisevi) sakupljeni su od 148 goveda tokom epizootije BKK na epizootiološkom području VSI Niš. Ukupno je ispitano je 167 uzoraka od 82 životinje sa kliničkim simptomima i 66 uzoraka krvi od 66 goveda bez kliničkih simptoma koji su uzorkovani pre vakcinacije, kao i 40 uzoraka poreklom od 20 goveda kod kojih su se klinički simptomi pojavili nakon vakcinacije. Iz uzorka bioptata kože goveda sa ispoljenim kliničkim simptomima urađena je izolacija virusa bolesti kvrgave kože. Izolovani virus (Pčinj 1) je identifikovan primenom klasičnih virusoloških (VNT) i molekularnih metoda (real-time PCR). Sekvenciranje delova genoma virusa BKK i to četiri gena: RPO30, GPCR, EEV i P32 urađeno je iz izolovanog virusa i šest uzoraka bioptata kože (pozitivnih real-time PCR) metodom po Sanger-u. Filogenetska analiza na osnovu dobijenih sekvenci RPO30, GPCR, EEV i P32 gena pokazala je potpunu podudarnost u redosledu nukleotida izolovanog i šest identifikovanih virusa BKK, kao i potpunu podudarnost sa analognim sekvencama soja Serbia/Bujanovac/2016. Utvrđeno je da oba soja virusa izolovana na području R. Srbije (Serbia/Bujanovac/2016 i Pčinj 1), kao i virusi detektovani u bioptatima kože pripadaju podgrupi terenskih sojeva virusa BKK. Analizom filogenetskog stabla formiranog na osnovu poređenja celog genoma soja Serbia/Bujanovac/2016 sa celim genomima 19 CaPV koji se nalaze u banci gena (GenBank) utvrđeno je da se soj Serbia/Bujanovac/2016 nalazi u podgrupi sa terenskim sojevima virusa BKK, pri čemu je najveća podudarnost u redosledu nukleotida utvrđena sa sojevima izolovanim u Grčkoj, Izraelu, Ruskoj Federaciji i Južnoj Africi. Dizajniranje sekvenci prajmera i probe za novi real-time PCR protokol za detekciju terenskog soja virusa (Terenski Niš) zasnovano je na razlikama u redosledu nukleotida na LSD008 genu između terenskih i vakcinalnih sojeva, dok je dizajniranje sekvenci prajmera i probe za novi realtime PCR protokol za detekciju vakcinalnog soja virusa (Vakcinalni Niš) zasnovano na razlikama u redosledu nukleotida na genu LSD146 terenskog i vakcinalnog soja virusa BKK. Ispitivanjem analitičke specifičnosti utvrđeno je da se protokolom Terenski Niš pored virusa BKK detektuju i virusi boginja i ovaca, ali se jasno diferenciraju terenski i vakcinalni soj virusa BKK. Real-time PCR protokol Vakcinalni Niš je specifičan samo za vakcinalne (Neethling) sojeve virusa BKK. Ispitivanjem analitičkih i dijagnostičkih performansi potvrđeno je da realtime PCR protokoli Terenski Niš i Vakcinalni Niš poseduju zadovoljavajuće performanse i mogu se koristiti u dijagnostici BKK. Ispitivanje prisustva genoma virusa BKK iz uzoraka izvršeno je primenom šest protokola za izvođenje real-time PCR metode (Bowden i sar., 2008, KV-2, Terenski Niš, KV-vac, Vakcinalni Niš i komercijalnim real-time PCR) i protokolom za izvođenje nested PCR metode (Menasherow i sar., 2014)...registered in the territory of the Republic of Serbia in June 2016. The aim of this doctoral dissertation was the isolation and identification of LSD virus, sequencing part of the genome of the isolated and identified LSD virus and molecular epizootiological analysis of the obtained data, as well as preparation and validation of new protocols for real-time PCR method to detect field and vaccine strain and define the most specific and sensitive protocol. An epizootiological analysis of the LSD epizootic that occurred in the epizootiological area controlled by the Veterinary Specialized Institute Nis in 2016, was also conducted within this doctoral dissertation. Samples for testing (blood, skin biopsies and nasal swabs from LSD suspected or susceptible cattle) were collected from 148 animals during the LSD epizootic in the epizootiological area of VSI Nis. A total of 167 samples from 82 animals with clinical symptoms and 66 blood samples from 66 animals without clinical symptoms that were sampled before vaccination, as well as 40 samples 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 that originated from animals with clinical symptoms. The isolated virus (Pčinj 1) was identified using classical virological (VNT) and molecular diagnostic methods (real-time PCR). Sequencing of parts of the LSDV genome, namely four genes: RPO30, GPCR, EEV and P32, was performed from the isolated virus and six skin biopsy samples (positive real-time PCR) by the Sanger method. Phylogenetic analysis based on the obtained sequences of RPO30, GPCR, EEV and P32 genes showed complete match in the sequence of nucleotides from isolated and six identified LSD viruses, as well as with analogous sequences of the Serbia/Bujanovac/2016 LSDV strain. It was 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 the subgroup of field strains of LSDV. Analysis of the phylogenetic tree formed on the basis of comparison of the whole genome of the strain Serbia/Bujanovac/2016 with the whole genomes of 19 CaPV located in the gene bank (GenBank) showed that the Serbia/Bujanovac/2016 strain is in a subgroup along with field strains of LSDV and has the highest nucleotide sequence match with 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 virus strain detection (Terenski Niš) was based on differences in the nucleotide sequence of the LSD008 gene between field and vaccine strains, while the design of primer and probe sequences for the new vaccine strain detection protocol (Vakcinalni Nis) was based on differences in the sequence of nucleotides on the LSD146 gene from both field and vaccine LSDV strains. By examining the analytical specificity, it was determined that the Terenski Niš protocol detects Goatpox and Sheeppox viruses in addition to the LSDV, but clearly differentiates the field and vaccine 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 the real-time PCR protocols Terenski Niš and Vakcinalni Nis have satisfactory performance and can be used in the diagnosis of LSD. Examination for the presence of LSDV genomes from the samples was performed using six different 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)..