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)..

Savremena arhitektonska praksa - institucionalni okvir i pravila građenja

Building rules represent important element of the system of planning in Serbia. It connects different kinds and scales of professional activities related to built environment, in first the larger scale of planning with smaller scales of designing and construction. The way the building rules are formulated, interpreted and applied in the procedures of planning, designing and construction the most directly influences the quality of the built and living environment. In this paper the professional experiences of the immediate participants in these procedures, previously exposed at the professional meeting entitled 'Contemporary architectural practice: institutional framework and building rules', are excerpted and systematized. The main goal is to point out to the problems in the current practice and to call upon the wider debate. The most important outcomes are the common attitudes as the basis for further dicussion and activities which would lead to the improvement of professional practice.Pravila građenja predstavljaju važan element sistema planiranja u Srbiji, koji povezuje različite oblike i razmere stručnih intervencija u prostoru, u prvom redu opštiji nivo planiranja sa konkretnijim nivoima projektovanja i izgradnje. Način na koji se formulišu, tumače i primenjuju pravila građenja u procedurama planiranja, projektovanja i građenja najdirektnije se odražava na kvalitet izgrađenog i životnog okruženja. U radu su izdvojena i sistematizovana profesionalna iskustva neposrednih učesnika u ovim procedurama, koja su prethodno izložena na tribini sa temom: Savremena arhitektonska praksa - institucionalni okvir i pravila građenja. Cilj je da se ukaže na probleme koji se javljaju u aktuelnoj praksi i podstakne šira debata. Rezultat tribine su zajednički stavovi koji predstavljaju osnovu za dalju diskusiju i delovanje u pravcu unapređenja profesionalne prakse

Contemporary practice of presentation: new tendencies in showing the cultural heritage

Zbog svog simboličkog značenja, kulturno-povijesno naslijeđe ima posebnu vrijednost za sadašnje naraštaje, utječe na formiranje identiteta i uobličavanje kolektivne i individualne kulture sjećanja. Veoma je važno da što više mladih ljudi dođe u dodir s umjetničkim djelima koja čine materijalno kulturno naslijeđe. Međutim, tradicionalni načini izlaganja često izazivaju otpor kod publike, već naviknute na dinamičnost novih medija. Suvremene izložbe postaju način da se publika informira, zabavi i obrazuje, one pričaju priče i nude argumente o raznovrsnosti i značaju kulturnog naslijeđa. Prednosti upotrebe nove tehnologije u prikazivanju i promociji kulturnog naslijeđa u ovom radu analizirat ćemo na primjeru multimedijalne izložbe „Osvježavanje memorije“. Kroz animaciju, projekcije i interaktivne prezentacije, intrigantno osvjetljenje i zvučne senzacije autor izložbe Dušan Milovanović i autor postava izložbe Ivan Mangov približili su, prije svega mlađoj publici, srpsko srednjovjekovno slikarstvo.Because of its symbolic meaning, the cultural and historical heritage has a specific value for the modern generations, since it forms the identity and shapes the collective and individual culture of memory. It is of great importance that more and more young people get in touch with the works of art that make the material cultural heritage. However, the traditional ways of presentation are usually not very popular with the audience, since people are so used to the dynamics of the new media. Contemporary exhibitions are becoming a way for the audience to get information, entertainment and education, since they tell stories and offer arguments of diversity and importance of cultural heritage. In this paper we will analyze the advantages of technology in presentation and promotion of cultural heritage on the example of the multimedia exhibition “Refreshing of memory”. Through animation, projection and interactive presentation, intriguing lighting and sound sensations, the author of the exhibition Ivan Mangov has presented the medieval painting to the younger audience

The Art of Spectacle as a Second Reality

Geneza medija kao višesmislenog fenomena podrazumeva njegov preobražaj od prvobitne funkcije odašiljača poruke i informacija u aktivnog subjekta u procesu komunikacije i moćnog sredstva kritičkog mišljenja. Na tom putu, mediji su dostigli snagu vodoćeg faktora u oblikovanju kritičkog mišljenja recipijenta. U radu se analiziraju komunikacijski procesi u savremenom društvu otkrivajući dominantan osećaj pasivne zavisnosti o medijske sadržaje od strane recipijenta i aktivne uloge medija. Tokom tog procesa informacija postaje antipodni entitet koji ukikida smisao i primarno služi kao podsećanje na neprikosnoveni kredibilitet koji je prenesen na spektakl kao umetnički izraz. U tom procesu recipijenta koji treba kritički da promišlja umetničke sadržaje preobražava se u nemoćnog i anestiziranog konzumenta svega što mu se predočava u medijima. Umetnost spektakla postaje realnost.A media genesis as multimeaningfull phenomenon means its transformation from its initial function as a transmiter of a message and information into an active subject in the process of communication towards a powerful mean of critical opinion. On that road, from the role of information transmition, medias strengtened into a leading factor in shaping critical opinion of a recipient. A project is focused on a communication processes analysis within modern society, revealing dominant feeling of passive dependency of a media content by the recipient and media’s active role. During that process, information becomes antipodal entity, which abolishes the meaning and primarily serves as a reminder on an undisputed credibility transfered into a spectacle as an art expression. Within that process, recipient who is supposed to criticaly think about media content transforms into a muted and brainwashed consumer of everything that is presented by media. The art of spectacle becomes reality

Seroprevalence nekih zaraznih bolesti u pasa lutalica u širem području Leskovca

Since the last decade, stray dogs have been significant ecological, economical and sociological problem in the world as well as in the Republic of Serbia. They occupy specifi c place in the ecology of big cities and their contact with human population represents danger since they are carriers of many zoonotic infectious diseases. The goal of this paper is to present epizootiological survey on some diseases of bacterial, viral and parasitic origin through serological investigation of blood sera and full blood. The prevalence of leptospirosis, Tularaemia, West Nile fever and dirofilariosis has been determined in this research. The material included full blood and blood sera of stray dogs in the wider territory of Leskovac city. The research methods encompassed standard serological tests: microscopic agglutination test (MAT) for determination of specific antibodies against Leptospira spp.; slow and fast agglutination test for determination of antibodies against Tularaemia causative agent (Francisella tularensis); agar gel immunodiffusion (AGID) for detection of specific antibodies against West Nile virus -WNV; modified Knot's test for the detection of microfilaria. The results revealed seropositivity for causative agents of Leptospirosis, Tularaemia and dirofilariosis, while there were no positive findings for WNV.Tokom poslednjih deset godina, psi lutalice se smatraju značajnim ekološkim, ekonomskim i socijalnim problemom kako u celom svetu tako i u Republici Srbiji. Psi lutalice zauzimaju posebno mesto u ekologiji velikih gradova i njihov kontakt sa ljudima, predstavlja opasnost, jer oni mogu biti nosioci uzročnika mnogih zoonotskih infektivnih oboljenja. Cilj ovog rada je da se predstavi epizootiološka studija nekih bakterijskih, virusnih i parazitskih oboljenja, pomoću seroloških ispitivanja krvnih seruma i pune krvi pasa. Tokom ovog istraživanja, ispitivana je prevalenca na leptospirozu, tularemiju, groznicu zapadnog Nila i dirofilariozu. Materijal su predstavljali uzorci pune krvi i krvnog seruma pasa lutalica, iz šire okoline grada Leskovca. Metode ispitivanja su obuhvatale standardne serološke testove: test mikroskopske aglutinacije (MAT) za utvrđivanje specifičnih antitela protiv Leptospira spp.; spora i brza aglutinacija za utvrđivanje antitela protiv uzročnika tularaemije (Francisella tularensis); agar gel imunodifuzioni test (AGID) za detekciju specifičnih antitela protiv virusa Zapadnog Nila -WNV; modifikovani Knotov test za detekciju mikrofilarija. Rezultati ispitivanja pokazuju seropozitivnost na uzročnike leptospiroze, tularemije i dirofilarioze, dok nisu dobijeni pozitivni nalazi za WNV. PR Projekat Ministarstva nauke Republike Srbije, br. TR 37015

Twenty-five-year study of Nosema spp. in honey bees (Apis mellifera) in Serbia

A total of 7386 samples of adult honey bees from different areas of Serbia (fifteen regions and 79 municipalities) were selected for light microscopy analysis for Nosema species during 1992-2017. A selection of honey bee samples from colonies positive for microsporidian spores during 2009-2011, 2015 and 2017 were then subjected to molecular diagnosis by multiplex PCR using specific primers for a region of the 16S rRNA gene of Nosema species. The prevalence of microsporidian spore-positive bee colonies ranged between 14.4% in 2013 and 65.4% in 1992. PCR results show that Nosema ceranae is not the only Nosema species to infect honey bees in Serbia. Mixed N. apis/N. ceranae infections were detected in the two honey bee samples examined by mPCR during 2017. The beekeeping management of disease prevention, such as replacement of combs and queens and hygienic handling of colonies are useful in the prevention of Nosema infection

Upcycling of the Used Cigarette Butt Filters through Pyrolysis Process: Detailed Kinetic Mechanism with Bio-Char Characterization

Thermo-chemical conversion via the pyrolysis of cigarette butt (CB) filters was successfully valorized and upcycled in the pre-carbonization and carbonization stages. The pre-carbonization stage (devolatilization) of the precursor material (cellulose acetate filter, r-CAcF) was analyzed by micro-scale experiments under non-isothermal conditions using TG-DTG-DTA and DSC techniques. The results of a detailed kinetic study showed that the decomposition of r-CAcF takes place via complex mechanisms, including consecutive reaction steps and two single-step reactions. Consecutive stages include the α-transition referred to as a cellulose polymorphic transformation (cellulose I → II) through crystallization mechanism changes, where a more thermodynamically ordered system was obtained. It was found that the transformation rate of cellulose I → II (‘cellulose regeneration’) is strongly affected by the presence of alkali metals and the deacetylation process. Two single-step reactions showed significant overlapping behavior, which involves a nucleation-controlled scission mechanism (producing levoglucosan, gaseous products, and abundant radicals) and hydrolytic decomposition of cellulose by catalytic cleavage of glycosidic bonds with the presence of an acidic catalyst. A macro-scale experiment showed that the operating temperature and heating rate had the most notable effects on the total surface area of the manufactured carbon. A substantial degree of mesoporosity with a median pore radius of 3.1695 nm was identified. The presence of macroporosity on the carbon surface and acidic surface functional groups was observed

Seroepidemiological survey of leptospiral infection in stray dogs in Serbia

Leptospirosis is a bacterial zoonosis with worldwide distribution. This disease is facilitated among stray dogs due to their lifestyle and the absence of immunoprophylaxis. The aim of the present study was to provide serological data on the presence of certain serovars of Leptospira spp., which are assumed to circulate in the population of stray dogs in Serbia. During a period of 3 years (from April 2010 to June 2013), 1045 canine sera originating from 11 shelters were submitted to the laboratory of the Department of Infectious Animal Diseases and Diseases of Bees, Faculty of Veterinary Medicine, Belgrade. A microscopic agglutination test (MAT) was performed to detect antibodies to Leptospira (cutoff, 1: 100). The overall seroprevalence was 5.45% (57/1045) and the most prevalent Leptospira serovars were Icterohaemorrhagiae 33.3% (19/57), Pomona 29.8% (17/57), Canicola 14.0% (8/57), Grippotyphosa 3.5% (2/57), Bataviae 1.7 % (1/57), and Sejroe 1.7% (1/57). All dogs were seronegative for antibodies against serovars Australis and Bratislava. The results showed that stray dogs contribute to the spread and maintenance of Leptospira spp. in Serbia. Due to close contact with humans it is very important to improve the prevention of leptospirosis in dogs and support a One Health approach

Seroprevalence of ehrlichia canis infection in stray dogs from Serbia

Canine Monocytic Ehrlichiosis is a zoonotic bacterial disease with worldwide distribution. With regards to the population of stray dogs, the disease is facilitated due to their lifestyle and the lack of anti-parasitic protection. The aim of this study was to provide serological data on the presence of a specific Ehrlichia canis IgG antibodies in stray dogs, originating from 7 municipalities in Serbia. During the period from April 2013 to June 2014, 217 canine sera were submitted to the laboratory of the Department of Infectious Diseases of Animals and Bees, Faculty of Veterinary Medicine in Belgrade. An immunofluorescent antibody test (IFAT) was performed to detect antibodies to Ehrlichia canis (cut off, 1: 50). Seropositive dogs were found in 5 out of 7 counties with a seroprevalence varying from 3.57% to 20% and an overall seroprevalence of 11.06% (24/217). There was no statistically significant difference between the prevalence of infection and the host age or gender. Results showed that stray dogs contribute to maintaining and spreading of Ehrlichia canis in Serbia. Due to the close relationship between people and dogs, it is of great importance to constantly monitor and improve prevention of this disease

Seroepizootiološko-epidemiološka istraživanja i mapiranje infekcije Zapadnog Nila u Republici Srbiji

The disease caused by the West Nile virus (WNV) has been known since 1937 when it was described for the first time in Uganda. After spreading to Europe and the Middle East, the disease has changed its primary location. Today WN infection is a significant health problem in the world. As a result of the current epizootiological and epidemiological situation in Europe studies on the occurrence of WND were introduced in Serbia, also. The investigation on the presence of WNV antibodies was intensified in the period from 2008 to 2012. In this period a total of 3618 serum samples were tested from 18 localities (2736 animal sera from 8 different species and 882 human sera samples). The method applied was gel immunodiffusion and the representative samples were confirmed by the plaque reduction test (PRNT-90). Out of the total number of samples WNV antibodies were present in 3.97% horses, 0.93% dogs, 0.31% poultry and 1.36% man. In one horse serum sample there was a positive reaction with a positive control serum, thus indicating the presence of WNV antigen. The results have confirmed that WNV antibodies are present in 9 out of 18 tested locations in the Republic of Serbia. The percentage of seropositive samples varies from 0.42% in Pozarevac (horses and humans) up to 6.45% in Novi Pazar (dogs). Out of the investigated species the highest seropositivity was recorded in horses (3.97%), and lowest in poultry (0.31%). WNV is present and widespread in the Republic of Serbia, thus enabling distribution mapping.Bolest koju izaziva virus Zapadnog Nila (WNV) je poznata još od 1937. kada je po prvi put opisana u Ugandi. Posle širenja virusa u Evropu i na Bliski istok, bolest je promenila prvobitnu lokaciju. Danas je WN infekcija značajan zdravstveni problem u svetu. Kao rezultat trenutne epizootiološko-epidemiološke situacije u Evropi, uvedena su istraživanja prisustva WNV i u Srbiji. Istraživanja prisustva antitela za WNV intenzivirana su u periodu od 2008. do 2012. godine. U ovom periodu ukupno je ispitano 3618 seruma sa 18 lokaliteta (2736 seruma životinja, od 8 različitih vrsta i 882 seruma ljudi). Korišćen je metod imunodifuzije u gelu a reprezentativni uzorak potvrđen je testom neutralizacije plaka (PRNT- 90). Od ukupnog broja uzoraka na prisustvo antitela za WNV bilo je pozitivno 3,97% konja, 0,93% pasa, 0,31% domaće živine i 1,36% ljudi. U serumu jednog konja pojavila se pozitivna reakcija sa pozitivnim kontrolnim serumom, ukazujući na prisustvo antigena WNV. Dobijeni rezultati su potvrdili da su antitela za WNV prisutna u 9 od 18 ispitivanih lokacija u Republici Srbiji. Procenat seropozitivnih varira od 0.42% u Požarevcu (konji i ljudi) do 6,45% u Novom Pazaru (psi). Od ispitivanih vrsta najveća seropozitivnost je registrovana kod konja (3,97%), a najniža kod živine (0,31%). Raspoloživi podaci ukazuju da je WNV prisutan i značajno raširen u Republici Srbiji, što je omogućilo i mapiranje njegovog širenja