Molecular characterisation of Parainfluenza 3 virus strains of cattle isolated on the theritory of Republic of Serbia

Glavni cilj ove doktorske disertacije je da se izvrši identifikacija i molekularna karakterizacija izolovanih sojeva virusa parainfluence 3 (PI3) goveda sa teritorije Republike Srbije. Izvršena ispitivanja su obuhvatila izolaciju virusa parainfluence 3 iz nosnih briseva goveda na kulturi tkiva i identifikaciju izolovanih sojeva virusa metodom virusneutralizacije, direktne imunofluorescencije i njihovu molekularnu karakterizaciju metodom lanĉane reakcije polimeraze (RT PCR) uz korišćenje prajmera za konzervisane regione HN i F gena virusa PI3 goveda sa sekvenciranjem radi njihove genotipizacije i svrstavanja na filogenetsko stablo izolovanih sojeva pomenutog patogena. Filogenetska analiza izolovanih sojeva izvršena je na osnovu utvrĊivanja homologije i poreĊenja pozicije njihovih nukleotidnih sekvenci sa sekvencama registrovanih sojeva virusa u bazi gena. Na osnovu nukleotidne homologije i pozicije domaćih izolovanih sojeva virusa na filogenetskom stablu, izvršena je njihova genotipizacija. U navedenim ispitivanjima je korišćen referentni soj SF4 virusa PI3 goveda (izolovan u drţavi Tenesi ,SAD). Nazalni brisevi poreklom od 119 goveda razliĉitih rasnih i starosnih kategorija sa ispoljenim simptomima infekcije gornjih partija respiratornog trakta su ispitivani na prisustvo virusa PI3 metodom izolacije na ćelijskoj liniji MDBK (Madin Darby Bovine Kidney). Izolacija virusa vršena je posle najviše tri pasaţe suspektnog materijala na navedenoj ćelijskoj liniji do pojave citopatogenog efekta. Citopatogeni efekat je ustanovljen u ćelijskim linijama posle inokulacije 11 uzoraka ispitivanog materijala, dok je primenom metode virusneutralizacije i direktne imunofluorescencije ustanovljeno prisustvo virusa parainfluece 3 kod 6 inokulisanih ćelijskih linija. U ostalim ćelijskim linijama, pojedinaĉno inokulisanim sa po 5 uzoraka poreklom od ispitivanih nosnih briseva goveda nije dokazano prisustvo virusa parainfluence 3 primenom prethodno navedenih metoda...The main objective of this doctoral thesis was the identification and molecular characterization of isolated strains of parainfluenza 3 (PI3) from cattle on the territory of the Republic of Serbia. The investigations included isolation of parainfluenza 3 virus from nasal swabs from cattle in tissue culture and identification of isolated strains of the virus by method of virusneutralisation, direct imunoflurescense and their molecular caracterisation by method of polymerase chain reaction (RT-PCR) using primers from conserved regions of the HN and F genes of the virus PI3 cattle, with sequencing and genotyping of domestic strains by help of their inclusion on the phylogenetic tree of mentioned pathogens. Phylogenetic analysis of the isolated strains was carried out on the basis of determination of the homology of the nucleotide sequences with the sequences of strains of the virus registered in gene base NCBI . Genotyping of domestic strains was done on the basis of nucleotide homology and position of local isolated virus strains on the phylogenetic tree, of PI3 virus. In these experiments, reference strain SF4 of virus PI3 of cattle was used (isolated in Tennessee, USA). Nasal swabs originating from 119 cattle with symptoms of infection in upper parts of the respiratory tract were examined for the presence of the virus PI3 by method of isolation of virus on cell line MDBK (Madin Darby Bovine Kidney). Virus isolation was done after three passages of suspected material on cell line till the appearance of cytopathic effect. Cytopathic effect was detected in cell lines after inoculation of 11 tested samples, while applying the method of virusneutralisation and direct immunofluorescense, identification of the virus parainfluece 3 were conformed in 6 inoculated cell lines, but not confirmed in other 5 tested samples from nasal swabs of cattle..

Molecular analysis of enterolysin A and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Soj Enterococcus faecalis BGPT1-10P je izolovan iz domaćeg polutvrdog sira, poreklom sa Stare Planine. Rezultati pokazuju da soj BGPT1-10P sintetiše termolabilan bakteriocin, enterolizin A, sa širokim spektrom delovanja, uključujući patogene bakterije roda Listeria i Candida. EntL gen, odgovoran za sintezu ovog bakteriocina, je hromozomalno lokalizovan. Analiza nukleotidne sekvence entL gena kod prirodnog izolata En. faecalis BGPT1-10P je identična sa entL genom soja En. faecalis LMG 2333, koji je prethodno okarakterisan. Pokazana je jedinstvena sekvenca entL gena i njegove okoline, koju čine orf1, orf2 i orf3 geni, kao i scpE gen. Prvi put je kod prirodnog izolata okarakterisan scpE gen, koji kodira virulentni faktor stafopain peptidazu. Funkcionalna analiza entL gena je pokazala da je kompletna genetička informacija, neophodna za sintezu i aktivnost enterolizina A, sadržana u entL genu. Soj BGPT1-10P osim enterolizina, sintetiše i želatinazu i citolizin i sadrži set različitih virulentnih faktora. Pored toga, BGPT1-10P nosi ermB i tetM gene, odgovorne za rezistenciju na eritromicin i tetraciklin.Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively

Molecular analysis of enterolysin a and entL gene cluster from natural isolate Enterococcus faecalis BGPT1-10P

Strain Enterococcus faecalis BGPT1-10P was isolated from artisanal semi-hard homemade cheese from Stara Planina, Serbia. Results showed that BGPT1-10P synthesized a heat labile bacteriocin with a broad spectrum of activity, including Listeria and Candida species. Further analysis revealed that synthesized bacteriocin is enterolysin A. Moreover, the entL gene encoding enterolysin A was found to be located on the chromosome. The entL gene was cloned and sequenced. Analysis of nucleotide sequence showed that the entL gene in natural isolate En. faecalis BGPT1-10P is identical to that of the entL gene described previously in En. faecalis LMG 2333. Within the cloned DNA fragment containing the entL gene, four ORFs were detected. One of them was identified as the scpE gene, which encodes a virulent factor staphopain peptidase. Functional analysis of the entL gene showed that the complete genetic information necessary for the synthesis of enterolysin A were directly linked solely to it. Strain BGPT1-10P also synthesized gelatinase and citolysin, and contained a set of virulent factors. In addition, BGPT1-10P carries the ermB and tetM genes conferring the resistance to erythromycin and tetracycline, respectively. [Projekat Ministarstva nauke Republike Srbije, br. 173019

Epizootiološka situacija afričke kuge svinja u Evropi

African swine fever (ASF) is a viral disease of domestic pigs and wild boar. Due to the very serious socioeconomic consequences, the disease is one of the most important ones nowadays. African swine fever is an enzootic disease in many countries in Sub-Saharan Africa, in Sardinia, and Trans Caucasus countries. After its occurrence in Georgia in 2007, ASF spread to Armenia and Russian Federation, and in 2008. to Azerbaijan. Since then, its progressive moving toward the west has been recorded. Despite the number of undertaken preventive and control measures in the European Union (EU), ASF has been still spreading. During 2017, the disease has been reported in domestic pigs in Estonia, Italy-Sardinia, Latvia, Lithuania, Poland, Romania, and Ukraine. ASF cases in domestic pigs have also been reported in Moldova in 2017. The number of diagnosed cases in wild boar in 2017 is much higher than in domestic pigs. ASF outbreak in wild boar in the Czech Republic well describes the possible viral 'jump' into a new region. The source of infection hasn't been confirmed yet, but it is common that such leaps are due to either swill feeding or improperly disposal of food rather than to the animal movements. Since the lack of effective vaccine makes eradication even more difficult, the prevention of viral entry into the new areas is of the most importance. With the same aim, since 2011.the surveillance of ASF has been implemented in Serbia.Afrička kuga svinja (AKS) je virusna bolest domaćih i divljih svinja. Socioekonomske posledice ove bolesti svrstavaju je u najznačajnije bolesti današnjice. Afrička kuga svinja je enzootska bolest u mnogim zemljama južno od Sahare, na Sardiniji i Kavkazu. Pošto se pojavila 2007. godine u Gruziji, AKS se iste godine proširila na Jermeniju i Rusiju, a 2008. na Azerbejdžan. Od tada se beleži progresivno kretanje virusa ka zapadu. Uprkos svim preventivnim i kontrolnim merama koje se sprovode u Evropskoj uniji (EU), afrička kuga svinja se i dalje širi. Tokom 2017. godine kod domaćih svinja je dokazana u Estoniji, Italiji - Sardinija, Letoniji, Litvaniji, Poljskoj, Rumuniji i Ukrajini. Slučajevi AKS kod domaćih svinja u Moldaviji su takođe registrovani i u 2017. godini. Broj dijagnostikovanih slučajeva kod divljih svinja u 2017. je značajno veći u odnosu na broj slučajeva kod domaćih. Pojava AKS u Češkoj 2017. godine kod divljih svinja predstavlja veliki "skok" virusa u novo područje. Izvor infekcije još uvek nije potvrđen, ali je uobičajeno da se ovakve pojave dešavaju kao posledica hranjenja životinja ostacima hrane, a ne zbog kretanja životinja. Budući da je iskorenjivanje AKS veoma otežano u odsustvu efikasne vakcine, prevencija unosa virusa u nova područja je od najvećeg značaja. Sa tim ciljem, u Srbiji se od 2011. godine sprovodi nadzor kod divljih svinja na afričku kugu

Important bacterial diseases and their control in rainbow trout in Serbian aquaculture

Global freshwater fish production in aquaculture has grown rapidly in recent decades. This constant growth, involving novel forms of intensive aquaculture, has increased global movements of fish and boosted various anthropogenic stresses to aquatic ecosystems, so rainbow trout aquaculture has encountered the emergence and outbreaks of many bacterial diseases. Due to the need to effectively prevent and control disease outbreaks, vaccines have become an important technology in intensive trout aquaculture. In this review, the applications of specific vaccines against important bacterial diseases of rainbow trout in Serbian aquaculture are summarized

Comparative analysis of antimicrobial and proteolytic activity of lactic acid bacteria isolated from Zlatar cheese

Tradicionalni zlatarski sir pripada grupi belih, polutvrdih sireva proizvedenih u domaćinstvu. Sir se proizvodi od nepasterizovanog kravljeg mleka bez dodavanja bilo kakvih poznatih starter kultura. Ukupno je izolovano 253 Gram pozitivnih i katalaza negativnih bakterija mlečne kiseline (BMK). Rezultati su pokazali da 70 od 253 analiziranih izolata proizvodi antimikrobna jedinjenja poznatih kao bakteriocini. Većina izolata koji pripadaju rodovima Lactococcus i Enterococcus, kao i izolati vrsta Lactobacillus plantarum i Lb. brevis ne sintetišu ekstracelularne proteinaze. Nasuprot njima, izolati prodvrste Lb. paracasei subsp. paracasei pokazuju veoma dobru proteolitičku aktivnoist. Pokazano je da ne postoji korelacija između dobre proteolitičke i antimikrobne aktivnosti u većini izolata.Traditional artisan Zlatar cheese belongs to the group of white, semi hard home-made cheeses, which are produced from no pasteurized cow's milk, without addition of any known bacterial starter culture. In total, 253 Gram-positive and catalase negative lactic acid bacteria (LAB) were isolated. Results showed that 70 out of 253 analyzed isolates produced antimicrobial compounds known as bacteriocins. Most isolates from genera Lactococcus and Enterococcus, and isolates belonging to species Lactobacillus plantarum and Lb. brevis, do not synthesize extracellular proteinase. In contrast, isolates from subspecies Lb. paracasei subsp. paracasei showed very good proteolytic activity. It was observed that good proteolytic activity of isolates was not in correlation with their good antimicrobial activity in the most of isolates

Examination of antimicrobial potential in natural isolates of lactobacillus casei/paracasei group

Cilj ove studije je izučavanje antimikrobnog potencijala 52 prirodna izolata vrste L. casei/paracasei. Učestalost gena koji kodiraju BacSJ (bacSJ2-8/bacSJ2-8i genski klaster), acidocin 8912 (acdT), ABC-transporter (abcT) i pomoćni protein (acc) su takođe izučavani. Genski klaster bacSJ2-8/bacSJ2-8i prisutan je kod 49 (94.23%), a acdT kod 41 (78.85%) od 52 testirana soja. Četrdeset sojeva (76.92%) poseduje oba analizirana gena. Interesantno je da samo 17 sojeva (32.69%) koji poseduju bacSJ2-8/bacSJ2-8i genski klaster i/ili acdT gen proizvode bakteriocine. Soj L. paracasei BGNK1-62 poseduje bacSJ2-8/bacSJ2-8i genski klaster, ali ne proizvodi bakteriocin BacSJ što je verovatno posledica nedostatka abcT i acc gena. Nakon transformacije soja BGNK1-62 konstruktom pA2A koji poseduje abcT i acc gene ostvarena je proizvodnja bakteriocina BacSJ. Osim toga, utvrđeno je da soj L. paracasei BGGR2-66 proizvodi nov bakteriocin označen kao BacGR, koji je biohemijski okarakterisan, a određena je i njegova N-terminalna sekvenca.The aim of this study was to investigate the antimicrobial potential of 52 natural isolates of Lactobacillus casei/paracasei. The incidence of relevant genes encoding BacSJ (bacSJ2-8/bacSJ2-8i gene cluster), acidocin 8912 (acdT), ABC-transporter (abcT) and accessory protein (acc) was also studied. These genes were found to be widespread amongst the analyzed L. casei/paracasei strains. The bacSJ2-8/bacSJ2-8i gene cluster was present in 49 (94.23%) and acdT in 41 (78.85%) of the 52 tested strains. Forty of these strains (76.92%) harbored both analyzed genes. Interestingly, only 17 strains (32.69%) with the bacSJ2-8/bacSJ2-8i gene cluster and/or the acdT gene showed bacteriocin production. Strain L. paracasei BGNK1-62 contained the bacSJ2-8/bacSJ2-8i gene cluster, but did not produce bacteriocin BacSJ possibly due to absence of the abcT and acc genes. Hence, these genes were introduced into BGNK1-62 by transformation with constructed plasmid pA2A, after which BacSJ was produced. In addition, it was found that L. paracasei BGGR2-66 produced new bacteriocin designated as BacGR that was biochemically characterized and its N- terminal sequence was determined

Analysis of the lactic acid bacteria microflora in traditional Caucasus cow's milk cheeses

Iz tri ručno pravljena sira uzeta iz različitih domaćinstava smeštenih u regionu planine Kavkaz, izolovano je ukupno 157 bakterija mleč ne kiseline (LAB). Sirevi su pravljeni od kravljeg mleka bez dodatka starter kulture. Izolati LAB su okarakterisani fenotipskim i genotipskim testovima. Rezultati identifikacije LAB pokazuju da je u ispitivanim sirevima prisutno deset vrsta, kao što su: Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium i Enterococcus faecalis. Sojevi u okviru vrsta L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis i L. pseudomesenteroides su pokazivali dobru proteolitičku aktivnost.A total of 157 lactic acid bacteria (LAB) were isolated from three hand-made cheeses taken from different households in the region of the Caucasus Mountains. The cheeses were manufactured from cow's milk without the addition of a starter culture. The isolates of LAB were characterized by subjecting them to phenotypic and genotypic tests. The results of identification of LAB indicate that the examined cheeses contained 10 species, viz., Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus farciminis, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides subsp. mesenteroides, Leuconostoc pseudomesenteroides, Enterococcus faecium, and Enterococcus faecalis. The strains within the species L. plantarum, L. arizonensis, L. paraplantarum, L. farciminis, and L. pseudomesenteroides showed good proteolytic activity

Characterization of lactic acid bacteria isolated from artisanal Zlatar cheeses produced at two different geographical location

Iz belog polutvrdog zlatarskog sira, označenog kao BGNV, izolovan je osamdeset jedan soj bakterija mlečne kiseline (BMK). Sir je uzet iz jednog seoskog domaćinstva smeštenog na severnoj strani planine Zlatar. Zlatarski BGNV sir je napravljen od svežeg kravljeg mleka bez dodatka starter kulture. Svi izolati BMK su okarakterisani fenotipskim i genotipskim testovima. Identifikacija sojeva je rađena rep-PCR analizom sa (GTG)5 prajmerom i 16S rDNK sekvenciranjem. Najzastupljenije vrste u zlatarskom BGNV siru su bile Lactobacillus casei/paracasei (65.43%) i Enterococcus faecalis (29.63%. Dva fakultetivno anaerobna štapića su identifikovana kao Lactobacillus plantarum (2.47%), a dva obligatna heterofermentativna izolata BMK su identifikovani kao Lactobacillus parabuchneri (2.47%). Od svih 81 testiranih izolata, samo osam eneterokoka su bili proizvođači antimikrobnih komponenti. Četrnaest od 16 testiranih izolata laktobacila je pokazivalo srednju do vrlo dobru proteolitičku aktivnost. Svih 57 laktobacila iz zlatarskog BGNV sira veoma sporo grušaju mleko, ili ga uopšte ne grušaju. Međutim, tri izolata enterokoka, BGNV1-63, BGNV1-76 i BGNV1-80, su pokazivala vrlo dobru aktivnost u mleku i grušala su ga za 5 h. Ove enterokoke su pokazivale vrlo visoku proteolitičku aktivnost potpuno hidrolizirajući αs1- i κ- kazein nakon 3 h, a β-kazein nakon 30 min inkubacije. Pored toga, ova tri izolata enterokoka degradovala su želatin. Upoređujući dobijene rezultate sa onima prethodno dobijenim ispitivanjem BMK u drugom zlatarskom BGZLS siru, napravljenom takođe, od svežeg kravljeg mleka, može se zaključiti da je mikroflora BMK zlatraskog BGNV sira manje raznovrsna.Eighty-one strains of lactic acid bacteria (LAB) were isolated from white semi-hard homemade cheese, designated Zlatar BGNV, which was taken from household settled on Northern side of mountain Zlatar. The Zlatar BGNV cheese was manufactured from raw cow's milk without addition of the starter culture. All isolates of LAB were characterized by phenotypic and genotypic tests. Identification of strains was done by the repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) with (GTG)5 primer and 16S rDNA sequence analysis. The most present species in Zlatar BGNV cheese were Lactobacillus casei/paracasei (65.43%) and Enterococcus faecalis (29.63%). Two facultative heterofermentative rods were identified as Lactobacillus plantarum (2.47%), and two obligate hetrofermentative LAB isolates as Lactobacillus parabuchneri (2.47%). Among all 81 tested isolates, only eight enterococci were producers of antimicrobial compounds. Fourteen of 16 tested lactobacilli isolates showed medium to very good proteolytic activity. All 57 lactobacilli from the Zlatar BGNV cheese curdled milk very slowly or did not curdle milk at all. However, three isolates of enterococci, BGNV1-63, BGNV1-76 and BGNV1-80, showed very good activity in milk and curdled milk within 5 h. They showed very high proteolytic activity hydrolyzing completely αs1- and κ-casein after 3 h, and β-casein after 30 min of incubation. In addition, those three enterococcal isolates degraded gelatin. Comparing obtained results with those previously achieved in examination of LAB microflora in another Zlatar BGZLS cheese made also from raw cow's milk, it can be concluded that LAB microflora in the Zlatar BGNV cheese is less diverse

Profiles of virulence genes in enterotoxigenic Escherichia coli isolates from suckling piglets in Serbia

A total of 120 Escherichia coli (E. coli) strains from suckling piglets with diarrhoea and 30 E. coli strains from healthy piglets were tested for the presence of fimbrial and enterotoxin virulence genes. Out of the 120 isolates sampled from diarrheic piglets, 81 (67.5%) expressed one or more genes encoding virulence factors. Adhesin genes were detected in 52 (43.33%) out of 120 E. coli isolates, and the most common among them was F4 adhesin (33.33%). Genes encoding E. coli toxins were detected in 81 (67.5%) isolates. E. coli included in the study carried genes for one or more of the following toxins: STa, STb, LT and EAST1. The astA gene encoding EAST1 was the most prevalent and was identified in 72 (60%) E. coli isolates. EAST1 toxin was detected in 5 out of 30 isolates (16.7%) from healthy piglets. Among the 81 isolates expressing virulence genes, a total of 15 different combinations for fimbrial and toxin genes were found. The most common virulence pattern was F4/STb/LT/EAST1 detected in 23.45% of E. coli strains isolated from suckling piglets with diarrhoea. The results indicate that F4 adhesin and EAST1 toxin are the most common in E. coli isolates sampled from diarrhoeic suckling piglets in Serbia