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This thesis consists of seven published papers on free-living amoebae (FLA), members of Amoebozoa, Excavata: Heterolobosea, and Cercozoa, and covers three main topics: (i) FLA as potential fish pathogens, (ii) diversity and phylogeography of FLA, and (iii) FLA as hosts of prokaryotic organisms. Diverse methodological approaches were used including culture-dependent techniques for isolation and identification of free-living amoebae, molecular phylogenetics, fluorescent in situ hybridization, and transmission electron microscopy.

<p style="text-align: justify;">Plodovi jabuke su tokom čitave godine prisutni u ishrani beba, dece i odraslih ljudi zbog čega je kvalitet i zdravstevena bezbednost ovih namirnica od izuzetnog značaja. Veliki broj fitopatogenih gljiva su prouzrokovači bolesti jabuka međutim, među značajnim prouzrokovačima bolesti jabuka u skladi&scaron;tima spominju se gljive iz rodova Alternaria i Fusarium. Smatra se da su zdravstveno bezbedni plodovi jabuke pre svega zdravi plodovi, bez prisustva fitopatogenih gljiva i bez ostataka fungicida. Sve veća briga oko primene sintetičkih fungicida zbog njihove toksičnosti, razvoja rezistentnosti patogena na fungicide i &scaron;tetnih efekata na životnu sredinu i zdravlje ljudi, doveli su do potrebe za pronalaženjem alternative hemijskoj za&scaron;titi, a jedno od mogućih se ogleda upravo u primeni biotehnolo&scaron;kim postupkom proizvedenih agenasa. Budući da su visoki tro&scaron;kovi biotehnolo&scaron;ke proizvodnje osnovna prepreka za &scaron;iru primenu ovih agenasa, optimizacija uslova izvođenja biosinteze predstavlja najvažniji korak ka njenom prevazilaženju. Stoga je kao cilj istraživanja u okviru ove doktorske disertacije definisana optimizacija biosinteze antagonista skladi&scaron;nih patogena jabuke primenom Streptomyces hygroscopicus na nivou laboratorijskog bioreaktora u pogledu odabira izvora ugljenika i azota i količina izvora ugljenika, azota i fosfora u hranljivoj podlozi za biosintezu željenih antagonističkih agenasa, kao i parametara biosinteze sa posebnim osvrtom na intenzitet primenjene aeracije i me&scaron;anja. Analizom dobijenih rezultata zaključeno je da se podloga za kultivaciju S. hygroscopicus koja je sadržala glicerol i pored već prisutnog azota u podlozi nije sadržala dodatni izvor azota, pokazala kao najpogodnija za produkciju željenih antagonističkih agenasa efikasnih protiv izolata vrsta A. alternata i F. avenaceum. Rezultati optimizacije sastava hranljive podloge ukazuju da se primenom definisanih optimalnih vrednosti ispitivanih faktora, koje su: sadržaj glicerola od 20 g/l, sadržaj (NH₄)₂SO₄ od 0,25 g/l i sadržaj K₂HPO₄ od 1,41 g/l u podlozi za biosintezu antagonista skladi&scaron;nih patogena jabuke primenom S. hygroscopicus, na osnovu definisanih matematičkih modela predviđaju sledeće vrednosti parametara toka i parametara uspe&scaron;nosti biosinteze antagonista skladi&scaron;nih patogena jabuke: rezidualni glicerol od 5,3418 g/l; rezidualni azot od 0,193 g/l; rezidualni fosfati od 0,8601 g/l; prečnik zone inhibicije rasta micelije izolata A. alternata KA10 od 28,22 mm; prečnik zone inhibicije rasta micelije izolata A. alternata T1Jg3 od 36,86 mm; prečnik zone inhibicije rasta micelije izolata F. avenaceum KA12 od 25,54 mm i prečnik zone inhibicije rasta micelije izolata F. avenaceum KA13 od 26,78 mm. Rezultati dobijeni ispitivanjem uticaja različitog intenziteta aeracije i brzine me&scaron;anja na biosintezu antagonista skladi&scaron;nih patogena jabuke primenom S. hygroscopicus u laboratorijskom bioreaktoru standardnih geometrijskih odnosa ukupne zapremine 3 l ukazuju da se primenom definisanih optimalnih vrednosti ispitivanih faktora, brzine me&scaron;anja od 100 o/min, intenziteta aeracije od 1,2 l/l/min i pri trajanju kultivacije od 96 h na osnovu definisanih matematičkih modela predviđaju sledeće vrednosti parametara toka i parametara uspe&scaron;nosti biosinteze antagonista skladi&scaron;nih patogena jabuke: rezidualni glicerol od 8,2645 g/l; rezidualni azot od 0,1723 g/l; rezidualni fosfati od 0,5167 g/l; suva materija 1,5023 g/l; prečnik zone inhibicije rasta micelije izolata A. alternata KA10 od 62,62 mm; prečnik zone inhibicije rasta micelije izolata A. alternata T1Jg3 od 61,09 mm; prečnik zone inhibicije rasta micelije izolata F. avenaceum KA12 od 45,55 mm i prečnik zone inhibicije rasta micelije izolata F. avenaceum KA13 od 43,02 mm. Validnost dobijenih rezultata potvrđena je izvođenjem biosinteze antagonista skladi&scaron;nih patogena jabuke na podlozi optimalnog sastava i primenom optimalnih procesnih parametara u laboratorijskom bioreaktoru standardnih geometrijskih odnosa ukupne zapremine 7l, a efikasnost produkovanih antagonističkih agenasa efikasnih na izolate A. alternata (KA10 i T1Jg3) i F. avenaceum (KA12 i KA13) potvrđena je i u in planta ogledima. Ostavreni rezultati optimizacije biosinteze antagonista skladi&scaron;nih patogena jabuke primenom Streptomyces hygroscopicus na nivou laboratorijskog bioreaktora predstavljaju osnovu za dalje uvećanje razmera do industrijskih, &scaron;to je ključni korak ka potencijalnoj industrijalizaciji predloženog proizvodnog postupka.</p> / <p>Apple fruits are available on the market all year round and their safety is of great importance for the consumers. Unfortunately, many phytopathogenic fungi can cause apple diseases but fungi from the genera Alternaria and Fusarium are listed among important storage pathogens of apple fruits. It is considered that health-safe apple fruits are primarily healthy fruits, without the presence of phytopathogenic fungi and without the remains of fungicides. Excessive use of pesticides in modern agriculture, including those used for control of fungal phytopathogens, has led to several problems related to pollution, environmental degradation and emergence of resistant strains. Biological<br />control of plant pathogens by means of microorganisms is considered as an attractive alternative to chemical-based treatments, with minimal impact on the environment. The main lack of biotechnology production of the antifungal agents are the high costs, so the optimization of the biosynthesis conditions is the most important step towards its overcoming. Therefore, the aim of this research is to optimize the medium composition and process conditions (agitation and aeration rate) of the antagonists of storage apple pathogens biosinthesis by Streptomyces hygroscopicus in laboratory-scale bioreactor. Based on the obtained results, it can be concluded that the S. hygroscopicus cultivation medium containing glycerol as a carbon source, with no additional nitrogen source, proved to be the most suitable for the production of the A. alternate and F. avenaceum antagonistic agents. The results of the optimization of medium for biosynthesis of the antagonists of storage apple pathogens using S. hygroscopicus indicated the following optimal values of the examined factors: the content of glycerol of 20 g/l, (NH₄)₂SO₄ of 0.25 g/l and K₂HPO₄ of 1.41 g/l. By applying the defined optimal values and using the appropriate mathematical models, the following responses are predicted: residual glycerol of 5.3418 g/l; residual nitrogen of 0.193 g/l; residual phosphates of 0.8601 g/l; inhibition zone diameters of A. alternata KA10 of 28.22 mm; inhibition zone diameters of A. alternata T1Jg3 of 36.86 mm; inhibition zone diameters of F. avenaceum KA12 of 25.54 mm and inhibition zone diameters of F. avenaceum KA13 of 26.78 mm. Examining the effect of different agitation and aeration rate on the biosynthesis of antagonists of storage apple pathogens using S. hygroscopicus in a laboratory-scale bioreactor with total volume of 3 l, the following optimal values of the investigated factors are obtained: the agitation rate of 100 rpm, the aeration of 1.2 l/l/min and 96-hour as optimal time of cultivation. Bringing together the appropriate mathematical models and defined optimal values, the following responses are predicted: residual glycerol of 8.2645 g/l; residual nitrogen of 0.1723 g/l; residual phosphates of 0.5167 g/l; biomass content of 1.5023 g/l; inhibition zone diameters of A. alternata KA10 of 62.62 mm; inhibition zone diameters of A. alternata T1Jg3 of 61.09 mm; inhibition zone diameters of F. avenaceum KA12 of 45.55 mm and inhibition zone diameters of F. avenaceum KA13 of 43.02 mm. The validity of the results was confirmed by performing the biosynthesis of the antagonists of storage apple pathogens in the medium with optimal composition and the optimal process parameters in a laboratory-scale bioreactor with the total volume of 7l. The efficacy of produced antagonistic agents effective against A. alternata (KA10 and T1Jg3) and F. avenaceum (KA12 and KA13) isolates was confirmed in vitro and in planta. The results obtained within this research represents the basis for the further enlargement of the scale, which is a key step towards the potential industrialization of the proposed production process.</p>

<p>Kontrola zdravlja vimena krava je bitan element u procesu proizvodnje zdravstveno<br />bezbednog mleka, te se na farmama visokomlečnih krava, kroz program kontrole mastitisa,<br />redovno sprovode mere otkrivanja i prevencije bolesti vimena. Klinički pregled vimena<br />predstavlja osnovni metod koji pruţa korisne informacije o zdravstvenom statusu vimena<br />krava, ali nailazi na pote&scaron;koće u otkrivanju patolo&scaron;kih promena unutar parenhima i papile<br />vimena. U cilju otkrivanja promena u parenhimu vimena moţe se primeniti ultrazvučni<br />pregled koji omogućava vizualizaciju strukturnih promena vimena nastalih kao posledica<br />upalnih procesa i tako olak&scaron;ava dijagnostiku oboljenja.<br />Tokom poslednjih godina, javio se problem povećanja rezistencije bakterija na<br />antimikrobne lekove, &scaron;to oteţava lečenje bolesti, ali i ugroţava zdravlje ţivotinja i ljudi.<br />Najče&scaron;ći uzroci toga su nepravilna upotreba i eventualna zloupotreba antimikrobnih lekova.<br />Mnoga istraţivanja vr&scaron;ena su u in vitro i in vivo uslovima na primeni laktoferina samog ili u<br />kombinaciji sa antibioticima u terapiji i prevenciji mastitisa krava. Laktoferin, gvoţĎe<br />vezujući antimikrobni glikoprotein koji se nalazi u mleku i drugim sekretima, predstavlja<br />bitan deo sistema odbrane mlečne ţlezde.<br />Cilj istraţivanja u okviru ove disertacije je procena dijagnostičke mogućnosti<br />ultrazvučnog pregleda u detekciji subkliničkog mastitisa i poremećene sekrecije vimena,<br />kao i razmatranje opravdanosti primene laktoferina u prevenciji i lečenju mastitisa.<br />Kliničkim pregledom izvr&scaron;ena je procena op&scaron;teg zdravstvenog stanja krava, kao i<br />ispitivanje mlečne ţlezde adspekcijom i palpacijom. Za otkrivanje poremećene sekrecije<br />vimena i subkliničkih mastitisa kori&scaron;ćeni su brzi testovi, Kalifornija mastitis test i<br />Draminski test, kao i ultrazvučni pregled mlečne ţlezde krava. OdreĎivanje broja somatskih<br />ćelija u uzorcima mleka uraĎeno je metodom protočne citometrije. Za identifikaciju<br />uzročnika mastitisa kori&scaron;ćene su klasične mikrobiolo&scaron;ke metode. Krave sa pozitivnim<br />bakteriolo&scaron;kim nalazom podeljene su u dve ogledne grupe. Krave ogledne grupe I su<br />tretirane intramamarnom aplikacijom antibiotika, dok je kravama ogledne grupe II<br />aplikovana kombinacija antibiotika i laktoferina. OdreĎivanje koncentracije imunoglobulina<br />G u mlečnom serumu krava vr&scaron;eno je metodom radioimunodifuzije, a odreĎivanje<br />koncentracije laktoferina u mleku krava ELISA testom.<br />U istraţivanjima u okviru ove disertacije, najče&scaron;će izolovani major mastitis patogeni<br />bile su bakterije Staphylococcus aureus i Streptococcus agalactiae, a najče&scaron;će izolovani<br />minor mastitis patogeni Corynebacterium spp. i koagulaza negativne stafilokoke.<br />Ultrasonografija mlečne ţlezde krava pokazala se kao efikasna metoda u dijagnostici<br />poremećaja sekrecije vimena. Veće vrednosti koncentracije imunoglobulina G u mlečnom<br />serumu krava uočene su tokom perioda predzasu&scaron;enja i zasu&scaron;enja, u odnosu na period rane<br />laktacije. Najveći uticaj na porast koncentracije laktoferina u mleku krava su imali<br />bakterijski uzročnici mastitisa. Efikasnost antibiotske terapije primenjene tokom perioda<br />zasu&scaron;enja kod krava ogledne grupe I iznosila je 52,7%, dok je efikasnost primenjene terapije<br />sa goveĎim laktoferinom i antibiotikom kod krava ogledne grupe II iznosila 60%. Aplikacija<br />laktoferina tokom perioda zasu&scaron;enja doprinela je efikasnosti terapije intramamarnih<br />infekcija, ali nije imala uticaj na koncentraciju laktoferina u mleku tokom perioda rane<br />laktacije.<br />Dalja istraţivanja su neophodna kako bi se u potpunosti potvrdila efikasnost<br />intramamarne terapije sa goveĎim laktoferinom i antibiotikom u cilju eliminacije uzročnika<br />mastitisa i da se precizno odredi terapijska koncentracija laktoferina.</p> / <p>Control of udder health is an essential element in the process of safe milk<br />production, thus, through the mastitis control program, dairy farms regularly conduct<br />measures of detection and prevention of udder diseases. Clinical examination of the udder<br />is a basic method that provides useful information about the health status of cow udders,<br />but this method is limited in its ability to detect pathological changes in the udder<br />parenchyma and teat. In order to detect changes in the parenchyma of the udder, an<br />ultrasound can be applied which allows visualization of udder structural changes incurred<br />as a result of inflammatory processes, thus facilitating diagnosis of diseases.<br />Over the last few years, the problem of increasing resistance to antimicrobial drugs<br />has appeared, making it difficult to treat disease and also threaten the health of animals and<br />humans. The incorrect and widespread use of antimicrobial drugs are the most common<br />cause of this. Many studies were conducted in vitro and in vivo conditions on the use of<br />lactoferrin alone or in combination with antibiotics in the treatment and prevention of<br />mastitis. Lactoferrin, an iron-binding antimicrobial glycoprotein which is found in milk<br />and other secretions, represents an important part of the mammary gland defense system.<br />The aim of the research within this thesis was to evaluate the diagnostic<br />possibilities of udder ultrasonography in the detection of subclinical mastitis and udder<br />secretion disorders, as well as considering the feasibility of lactoferrin application in the<br />prevention and treatment of mastitis.<br />The general condition of the animals was evaluated by clinical examination, as<br />well as udder examination by adspection and palpation. The California mastitis test,<br />Draminski test and ultrasound examination of the cow&#39;s mammary glands were used for<br />detection of udder secretion disorders and subclinical mastitis. Somatic cell count in milk<br />samples was determined by flow cytometry method. Classical microbiological methods<br />were used for the isolation and identification of mastitis causative agents. Cows with<br />positive bacteriological findings were divided into two experimental groups. Cows in<br />experimental group I were treated with intramammary applications of antibiotics, while the<br />cows in experimental group II were treated with a combination of antibiotics and<br />lactoferrin. Immunoglobulin G concentration in bovine milk serum was determined by the<br />immunodiffusion method, while lactoferrin concentration in bovine milk was determined<br />using the ELISA test.<br />In this study, the most common isolated major mastitis pathogens were<br />Staphylococcus aureus and Streptococcus agalactiae, while Corynebacterium spp. and<br />coagulase-negatice staphilococci were the most commonly detected minor mastitis<br />pathogens. Ultrasonography of the bovine mammary gland proved to be an effective<br />method in the diagnosis of udder secretion disorders. Higher immunoglobulin G<br />concentrations were observed in milk serum from cows during pre-dry and dry period,<br />relative to early lactation period. The biggest influence on the increase in the concentration<br />of lactoferrin in the bovine milk had mastitis pathogens. The efficacy of antibiotic therapy<br />during the dry period in cows of experimental group I was 52.7%, while the efficacy of the<br />applied therapy with lactoferrina and antibiotics in cows of experimental group II was<br />60%. Application of lactoferrin during the dry period contributed to the effectiveness of<br />the treatment of intramammary infections, but had no influence on lactoferrin<br />concentration in the milk during the early lactation period.<br />Further studies are necessary to in order to fully confirm the efficacy of<br />intramammary therapy with bovine lactoferrin and antibiotic to eliminate the mastitis<br />pathogens and to determine the therapeutic concentration of lactoferrin.</p>