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Enterokoke pripadaju grupi mleĉnokiselinskih bakterija koje su široko rasprostranjene u prirodi. Predominantno se nalaze u gastrointestinalnom traktu životinja, od insekata do ĉoveka i preko fecesa se oslobaĊaju u spoljašnju sredinu, nakon ĉega naseljavaju razliĉita staništa. Imaju kontroverzni status zbog uloge u bolniĉkim infekcijama i širenja gena odgovornih za rezistenciju na antibiotike. S druge strane, neki sojevi se koriste kao probiotici za životinje i ĉoveka, kao i starter kulture u mlekarskoj industriji. Da bi se enterokoke koristile kao probiotici neophodno je da budu bezbedne za korišćenje, da nemaju hemolitiĉku i želatinoznu aktivnost i da nisu otporne na antibiotike. Enterokoke sintetišu antimikrobne proteine, bakteriocine, i na taj naĉin regulišu brojnost drugih bakterija i modulišu odgovor domaćina na infekciju. One mogu da sintetišu i biomolekule koji interaguju sa ćelijama domaćina i na taj naĉin mogu da menjaju razliĉite signalne puteve unutar ćelija domaćina. Biomolekuli sintetisani od strane probiotiĉkih enterokoka nazivaju se postbiotici. Primena postbiotika predstavlja bezbednu alternativu korišćenja enterokoka u biomedicini. Ova doktorska disertacija imala ja za cilj da ispita probiotiĉka i imunomodulatorna svojstva prirodnih sojeva enterokoka izolovanih iz fermentisanih mleĉnih proizvoda sa podruĉja Zapadnog Balkana. Definisana su tri cilja: 1. da se pronaĊu sojevi bezbedni za korišćenje u biomedicini; 2. da se ispitaju probiotiĉke karakteristike tih sojeva u modelima in vitro; i 3. da se ispita imunomodulatorni efekat odabranog soja u modelima in vitro i in vivo. U cilju ispitivanja bezbedne upotrebe enterokoka kao probiotika testirano je ukupno 75 sojeva iz fermentisanih mleĉnih proizvoda koje pripadaju vrstama: Enterococcus durans (50 sojeva), Enterococcus faecium (15 sojeva), Enterococcus faecalis (6 sojeva), Enterococcus italicus (3 soja) i Enterococcus hirae (1 soj). Hemolitiĉku aktivnost pokazalo je 18,7% (14/75), dok je želatinaznu aktivnost imalo 6,7% (5/75) testiranih sojeva. Na osnovu rezultata testa mikrodilucije pokazana je visoka uĉestalost rezistencije na ciprofloksacin 48,2% (27/56), dok su u manjoj meri testirani sojevi rezistentni na gentamicin 10,7% (6/56). Na osnovu rezultata sposobnosti formiranja biofilma u primenjenim uslovima, devet sojeva nema sposobnost formiranja biofilma, 11 sojeva ima mogućnost formiranja slabog biofilma, BGGO9-28 ima sposobnost formiranja jakog biofilma, dok sojevi BGTRK4-42 i BGZLM1-5 pokazuju veoma jaku sposobnost formiranja... are predominantly found in the gastrointestinal tract of animals, from insects to humans and through faeces they are released into the environment, where they colonize different habitats. They have a controversial status according to the role as causative agents of hospital infections and the spread of genetic determinants of antibiotic resistance. In contrary, some strains have been used used as probiotics for animals and humans, as well as a starter cultures in the dairy industry. In order to use enterococci as probiotics, it is necessary to determine their safety, i.e. absence of hemolytic and gelatinase activity and susceptiblity to clinicaly relevant antibiotics. Enterococci synthesize antimicrobial proteins, bacteriocins, by which way they regulate the number of other bacteria and modulate the host response to the infection. They can also synthesize biomolecules that interact with the host cells, and change the different signalling pathways within host cells. Biomolecules synthesized by probiotic enterococci are called postbiotics. The use of postbiotic represents a safety alternative to the enterococci application in biomedicine. The aim of this dissertation was examination of probiotic and immunomodulatory characteristics of enterococci isolated from fermented dairy products from the Western Balkans countries. Three objectives were defined: 1. to find strains safe for use in biomedicine; 2. to examine the probiotic characteristics on in vitro models, and 3. to examine the immunomodulatory effect of the selected strain on in vitro and in vivo models. In order to analyse the safe use of enterococci as a probiotics, a total of 75 isolates from fermented dairy products were tested: Enterococcus durans (50 isolates), En. faecium (15 isolates), En. faecalis (6 isolates), En. italicus (3 isolates) and En. hirae (1 isolate). 18.7% (14/75) strains showed hemolytic activity, while 6.7% (5/75) had gelatinase activity. Based on the results of the microdilution test, 48.2% (27/56) of strains were ciprofloxacin resistance, while 10.7% (6/56) strains were resistant to gentamicin. According to the analysis of selected enterococci genomes, high incidence of adhesin encoding genes could be noticed. It was found that 30.4% of strains have three genes encoding different virulence factors, 21.7% of strains having two or four genes, 17.4% having five genes, and 4.3% of studied enterococci containing 6 or 7 genes which encode virulence factors, while the presence or absence of gene within the fsr operon varies from the analyzed strains...

To keep pace with competing farmers in terms of technological equipment, and thus the amount of yield and quality of those products, traditional farmers are forced to carry out the modernization of its technical and technological capacity. The development of a number of financial services in the past decade and their offerings by financial institutions in the Republic of Serbia, made available to interested parties numerous opportunities for funding the purchase of equipment. Respecting all the difficulties that local farmers have faced in the past, the paper will consider the possibility of using borrowed funding, primarily loans and leases, as well as alternatives and their impact on the competitiveness of users.

Salmonele predstavljaju veoma važne patogene mikroorganizme ljudi i životinja koji su odgovorni za 93,8 miliona infekcija i 155.000 smrtnih slučajeva ljudi godišnje u svetu. Uzročnik vedine slučajeva salmoneloze ljudi je hrana, ali su i brojne infekcije posledica direktnog ili indirektnog kontakta sa životinjama. Gmizavci predstavljaju značajni rezervoar velikog broja podvrsta i serovarijeteta Salmonella spp., a brojni radovi potvrđuju mogudnost prenošenja ovih bakterija sa gmizavaca na ljude. Cilj ove disertacije je bio da se ustanovi prisustvo i raširenost serovarijeteta Salmonella spp. i njihove rezistencije prema antimikrobnim sredstvima. Od aprila 2012. do aprila 2016. godine bakteriološki su ispitivani brisevi i feces 317 gmizavaca i vodozemaca, slobodnih u prirodi, držanih u zoološkom vrtu ili gajenih kao kudni ljubimci (152 kornjače, 76 guštera, 73 zmije i 16 žaba) poreklom od 42 različite vrste i izolovano je ukupno 57 sojeva Salmonella enterica (18,93%) od 301 gmizavca, za razliku od vodozemaca gde su svi uzorci bili negativni. Salmonella enterica je izolovana kod 24 kornjače (15,79%,), 14 guštera (18,42%) i 19 zmija (26,03%), a nakon serotipizacije 41 soj je identifikovan kao S. enterica subsp. enterica (I) i 16 kao S. enterica subsp. diarizonae (IIIb). Ukupno je serotipizacijom utvrđeno prisustvo 24 različita serovarijeteta Salmonella enterica, od kojih po prvi put u Srbiji 10 serovarijeteta, a jedan determinisani serovarijetet se ne nalazi u White-Kauffmann-Le Minor tabeli. Kod sojeva Salmonella nije utvrđena rezistencija prema ispitivanim antimikrobnim sredstvima. Salmonella Enteritidis, Salmonella Hadar i Salmonella Thompson koje su otkrivene kod gmizavaca spadaju u serovarijetete Salmonella koje dovode do infekcija ljudi. Dobijeni rezultati nedvosmisleno ukazuju da gmizavci, kako slobodni u prirodi tako i u zoološkom vrtu ili držani kao kudni ljubimci, mogu predstavljati rezervoar Salmonella spp., što je od velikog epidemiološkog značaja usled zoonoznog potencijala uzročnika. Otkrivanje retkih i egzotičnih serovarijeteta Salmonella spp. ukazuje na neophodnost sprovođenja daljih sveobuhvatnih istraživanja i sistematskog pristupa u razjašnjenju uloge gmizavaca u epidemiologiji salmoneloze.

Hemoparaziti iz familije Haemogregarinidae često parazitiraju kod reptila, a u našoj zemlji do sada ne postoje podaci o infekciji uzročnicima hemoparazitom Haemogregarina stepanowi, kod jedine zaštićene vrste kornjača - evropske barske kornjače (Emys orbicularis), ni u prirodnim staništima, ni u kontrolisanim uslovima (zoo vrtovi, karantin). Klinički simptomi infekcije kornjača ovim hemoparazitom su nespecifični, nedostaju podaci o potencijalnim vektorima i o tome kakvu opasnost oni nose za druge životinje i čoveka. Ispitivanjima je obuhvaćena populacija od trideset barskih kornjača poreklom iz prirodnih staništa, a privremeno smeštenih u karantinu Zoo vrta Beograd, sa simptomima opšte slabosti i otežanog kretanja. Šest zdravih barskih kornjača, služile su kao kontrolna grupa za kliničke i citološke analize kao i molekularno-genetička ispitivanja. Životinje su pažljivo makroskopski pregledane i markirane radi kasnije identifikacije. Nakon uzorkovanja krvi vraćene su na mesto gde su izlovljene u skladu sa čl.78. Zakona o zaštiti prirode (“Službeni glasnik RS”, br.5/10, 47/11). Za ovu vrstu ispitivanja je dobijena dozvola – rešenje br. 119- 01-13-2-2015-09 od 12.01.2015. od Ministarstva poljoprivrede i zaštite životne sredine. Molekularno-genetičkim analizama (PCR) ustanovljeno je prisustvo Haemogregarina stepanowi u uzorcima krvi svih trideset odraslih kornjača oba pola. Molekularno-genetička ispitivanja uzoraka krvi urađena su u cilju identifikacije hemoparazita Haemogregarina stepanowi. Izolacija DNK iz parafinskih isečaka tkiva 5 μm i krvi ispitivanih kornjača rađena je primenom komercijalnog seta “KAPA Express Extract Kit” prema protokolu proizvođača. Ispitivanje prisustva nukleinske kiseline parazita Haemogregarina stepanowi urađeno je kod ukupno 36 uzoraka krvi barskih kornjača (Emys orbicularis). Primenom prajmera EF: (5’ – GAAACTGC- GAATGGCTCATT -3) i ER: (5’- CTTGCGCCTACTAGGCATTC-3’) amplifikovani su 1500 bp dugački fragmenti jedarne 18S rDNK. Nakon vizualizacije na 1,5% agaroznom gelu, kod 30 ispitivanih uzoraka amplifikati su bili prisutni. Nakon amplifikacije željenog segmenta DNK parazita, dobijeni amplifikati su iskorišćeni za sekvencioniranje željnog segmenta gena odgovornog za sintezu 18S ribozomalne DNK. Rezultati sekvencioniranja (Gen Bank Number KT749877) potvrdili su da su svi ispitivani uzorci 100% identični sekvencama 18S ribozomalne DNK, koje su prethodno prijavljene u genskoj bazi od strane drugih autora (KF257929. 1, KF257927.1)... Hemoparasites from the Haemogregarinidae family often parasite on reptiles, in our country there is no data on infection of the only protected turtle – European pond turtle (Emys orbicularis) caused by Haemogregarina stepanowi, neither in their natural habitat nor under controlled conditions (zoo gardens, quarantine). Clinical symptoms of the infection caused by this hemoparasite are not specific and there is a lack of information on potential vectors and on the risk they pose to other animals and humans. The investigations covered a population of thirty pond turtles originating from their natural habitat, temporarily placed in quarantine at the Belgrade Zoo, with symptoms of general weakness and difficulty in movement. Six healthy pond turtle, served as a control group for clinical and cytological analysis and molecular genetic testing. The animals were carefully examined macroscopically and marked for later identification. Six healthy pond turtles served as the control group for clinical and cytological analysis and molecular genetic testing. After blood sampling they were returned to the place where they were caught, in accordance to article 78, Nature Protection Act (“Official Gazette”, No.5 / 10, 47/11). For this type of testing in a permission has been obtained - No. 119-01-13-2-2015-09 dated 12.01.2015 by the Ministry of Agriculture and Environmental Protection. Molecular genetic analysis (PCR) established the presence of Haemogregarina Stepanowi in the blood samples of all thirty adult turtles of both sexes. Molecular - genetic tests of blood samples were carried out in order to identify Haemogregarina stepanowi. Isolation of DNA from tissue samples embedded in paraffin and measuring 5 microns blood samples tests was done using the commercial set “KAPA Express Extract kit” according to the manufacturer’s protocol. Detection of Haemogregarina Stepanowi nucleic acids was done on a total of 36 blood samples of the pond turtle (Emys orbicularis). By applying the primer EF: (5 ‘- GALACTIC- GAATGGCTCATT -3) and ER (5 ‘- CTTGCGCCTACTAGGCATTC-3’) amplified 1500 bp long fragments of the nuclear 18S rDNA. After visualization on a 1.5% agarose gel in the 30 tested samples amplified molecules were present. After amplification of the desired parasite DNA segments, the obtained amplified molecules were used for sequencing the desired segment of the gene responsible for the synthesis of 18S ribosomal DNA...

Kutani mastocitomi su jedna od najčešći neoplazmi pasa. Vode poreklo iz kostne srži, iz linije opredeljene za mastocite i pokazuju vrlo promenljivo ponašanje, te je predloženo nekoliko kriterijuma za nji ovu klasifikaciju, uključujući istološku gradaciju i markere proliferacije ćelija Citoplazmatska ekspresija CD117 ili KIT proteina (tirozin kinaza receptor) dovodi se u vezu sa višim stepenima maligniteta mastocitoma i lošijom prognozom Dokazano je da postoji veza između aberantne ekspresije CD117 i postojanja c-kit mutacija, iako mutacije često nisu prisutne kod svih mastocitoma sa aberantnom ekspresijom KIT proteina. Ispitivanjima su obu vaćena 24 kutana mastocitoma i 6 kontrolni uzoraka kože od 30 pasa različitog uzrasta, rase i pola. Kao pozitivna kontrola za c-kit mutaciju korišćen je izolat DNK psa sa mastocitomom za koga je ranije utvrđeno da poseduje navedenu mutaciju Uzorci tkiva fiksiranog u formalinu i ukalupljenog u parafinske blokove, bojeni su hematoksilin-eozinom i toluidin blue metodom, a imunohistohemijski na CD117, Ki-67, PCNA i CD45RA. DNK je izolovana iz istih parafinskih blokova, a primenom PCR metode i korišćenjem specifični prajmera PE1 i PE2 amplifikovan je željeni fragment DNK. Četrnaest od 24 mastocitoma su prema istološkim osobinama svrstana u mastocitome visokog stepena maligniteta, dok su preostalih deset, mastocitomi niskog stepena maligniteta. Kod 7 mastocitoma prisutno je više od šest mitotskih figura na 10 polja velikog uvećanja Prisustvo više od dve multinuklearne ćelije na 10 polja velikog uvećanja uočeno je kod 11 mastocitoma, dok su više od dve ćelije sa bizarnim jedrom na 10 polja velikog uvećanja uočene kod 10 mastocitoma. Osam mastocitoma sadrži više od 10% anaplastični mastocita sa jedrima promera oko 8-10 mikrometara. Od ukupno 24 mastocitoma, devet je Ki-67 pozitivno (7 mastocitomima višeg i 2 mastocitomima nižeg stepena maligniteta), dok se ekspresija PCNA i CD45RA uočava kod svih mastocitoma. Devet mastocitoma višeg stepena maligniteta ispoljava aberantnu citoplazmatsku ekspresiju CD117 Ni kod jednog od ispitanih uzoraka mastocitoma nije detektovano prisustvo c-kit mutacije. Prisustvo mitotskih figura, višejedarni ćelija, bizarnih jedara i kariomegalije, kao i tip ekspresije CD117 predstavljaju preduslov za novu klasifikaciju i ključne prognostičke parametre kod pasa sa mastocitomima. Cutaneous mast cell tumours (MCTs) are one of the most common neoplasm in dogs. Originate from the bone marrow, from the mast cells line, but because of its location are being studied within skin tumors. MCTs show a highly variable biologic behavior and therefore several criteria have been proposed for their classification, including histological grading and cell proliferation markers. Cytoplasmic expression of CD117 or KIT protein (tyrosine kinasa receptor) has been found to correlate with higher histological grade and with a worsened prognosis. It has been revealed that there is a conection between the aberrant expression of CD117 and presence of c-kit mutations, although mutations were not present in all mast cell tumors with aberrant expression of KIT protein. Twenty four MCTs and 6 control specimens from 30 dogs of different age, breed and sex were included in this study. DNA isolated from the dog with mast cell tumor which had previously found to has specified mutation was used as a positive control for c-kit mutation. Formalin fixed and paraffin embedded tissue samples were stained with hematoxylin-eosin and toluidin blue and immunohistochemically tested for CD117, Ki-67, PCNA i CD45RA expression. DNA had been extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Fourteen of 24 MCTs were histologically high-grade, while ten were classified as a low-grade malignancy. Seven MCTs have more than six mitotic figures in 10 high-power fields. The presence of more than two multinucleated cells in 10 high-power fields was observed in 11 MCTs, while more than two cells with bizarre nuclei in 10 hpf were observed in 10 MCTs. Eight MCTs have more than 10% of neoplastic mast cells with nuclei about 8-10 microns in diameter. Of the total MCTs, 9 tumors are Ki-67 positive (7 mastocytomas of high and 2 mastocytomas of low-grade malignancy), while the expression of PCNA and CD45RA was observed in all 24 MCTs. Nine MCTs of high-grade malignancy showed aberrant cytoplasm labeling for CD117. Mutations of c-kit proto-oncogene were not detected in any of the tested MCTs. The presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high-power fields, as well as the pattern of CD117 expression are prerequisite for the new classification and most important prognostic factor in dogs with mast cell tumor.

Cutaneous mast cell tumours (MCTs) are one of the most common neoplasm in dogs. Originate from the bone marrow, from the mast cells line, but because of its location are being studied within skin tumors. MCTs show a highly variable biologic behavior and therefore several criteria have been proposed for their classification, including histological grading and cell proliferation markers. Cytoplasmic expression of CD117 or KIT protein (tyrosine kinasa receptor) has been found to correlate with higher histological grade and with a worsened prognosis. It has been revealed that there is a conection between the aberrant expression of CD117 and presence of c-kit mutations, although mutations were not present in all mast cell tumors with aberrant expression of KIT protein. Twenty four MCTs and 6 control specimens from 30 dogs of different age, breed and sex were included in this study. DNA isolated from the dog with mast cell tumor which had previously found to has specified mutation was used as a positive control for c-kit mutation. Formalin fixed and paraffin embedded tissue samples were stained with hematoxylin-eosin and toluidin blue and immunohistochemically tested for CD117, Ki-67, PCNA i CD45RA expression. DNA had been extracted from the same paraffin blocks and subsequent polymerase chain reaction amplification was performed using PE1 and PE2 primers. Fourteen of 24 MCTs were histologically high-grade, while ten were classified as a low-grade malignancy. Seven MCTs have more than six mitotic figures in 10 high-power fields. The presence of more than two multinucleated cells in 10 high-power fields was observed in 11 MCTs, while more than two cells with bizarre nuclei in 10 hpf were observed in 10 MCTs. Eight MCTs have more than 10% of neoplastic mast cells with nuclei about 8-10 microns in diameter. Of the total MCTs, 9 tumors are Ki-67 positive (7 mastocytomas of high and 2 mastocytomas of low-grade malignancy), while the expression of PCNA and CD45RA was observed in all 24 MCTs. Nine MCTs of high-grade malignancy showed aberrant cytoplasm labeling for CD117. Mutations of c-kit proto-oncogene were not detected in any of the tested MCTs. The presence of mitotic figures, multinucleated cells, bizarre nuclei and karyomegaly in 10 high-power fields, as well as the pattern of CD117 expression are prerequisite for the new classification and most important prognostic factor in dogs with mast cell tumor. Kutani mastocitomi su jedna od najčešći neoplazmi pasa. Vode poreklo iz kostne srži, iz linije opredeljene za mastocite i pokazuju vrlo promenljivo ponašanje, te je predloženo nekoliko kriterijuma za nji ovu klasifikaciju, uključujući istološku gradaciju i markere proliferacije ćelija Citoplazmatska ekspresija CD117 ili KIT proteina (tirozin kinaza receptor) dovodi se u vezu sa višim stepenima maligniteta mastocitoma i lošijom prognozom Dokazano je da postoji veza između aberantne ekspresije CD117 i postojanja c-kit mutacija, iako mutacije često nisu prisutne kod svih mastocitoma sa aberantnom ekspresijom KIT proteina. Ispitivanjima su obu vaćena 24 kutana mastocitoma i 6 kontrolni uzoraka kože od 30 pasa različitog uzrasta, rase i pola. Kao pozitivna kontrola za c-kit mutaciju korišćen je izolat DNK psa sa mastocitomom za koga je ranije utvrđeno da poseduje navedenu mutaciju Uzorci tkiva fiksiranog u formalinu i ukalupljenog u parafinske blokove, bojeni su hematoksilin-eozinom i toluidin blue metodom, a imunohistohemijski na CD117, Ki-67, PCNA i CD45RA. DNK je izolovana iz istih parafinskih blokova, a primenom PCR metode i korišćenjem specifični prajmera PE1 i PE2 amplifikovan je željeni fragment DNK. Četrnaest od 24 mastocitoma su prema istološkim osobinama svrstana u mastocitome visokog stepena maligniteta, dok su preostalih deset, mastocitomi niskog stepena maligniteta. Kod 7 mastocitoma prisutno je više od šest mitotskih figura na 10 polja velikog uvećanja Prisustvo više od dve multinuklearne ćelije na 10 polja velikog uvećanja uočeno je kod 11 mastocitoma, dok su više od dve ćelije sa bizarnim jedrom na 10 polja velikog uvećanja uočene kod 10 mastocitoma. Osam mastocitoma sadrži više od 10% anaplastični mastocita sa jedrima promera oko 8-10 mikrometara. Od ukupno 24 mastocitoma, devet je Ki-67 pozitivno (7 mastocitomima višeg i 2 mastocitomima nižeg stepena maligniteta), dok se ekspresija PCNA i CD45RA uočava kod svih mastocitoma. Devet mastocitoma višeg stepena maligniteta ispoljava aberantnu citoplazmatsku ekspresiju CD117 Ni kod jednog od ispitanih uzoraka mastocitoma nije detektovano prisustvo c-kit mutacije. Prisustvo mitotskih figura, višejedarni ćelija, bizarnih jedara i kariomegalije, kao i tip ekspresije CD117 predstavljaju preduslov za novu klasifikaciju i ključne prognostičke parametre kod pasa sa mastocitomima.