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In September 2018, a four-month-old dog with fever and petechial bleeding came to the internal clinic at the Faculty of Veterinary Medicine University of Belgrade. On hematology analysis, thrombocytopenia and mild anemia were observed. Examination of the blood smear revealed platelet inclusions. The commercial serology test was positive for Anaplasma spp. The dog was treated with doxycycline for 14 days, and after 48 hours from the beginning of the treatment, the symptoms subsided. PCR analysis and sequencing confirmed infection with A. platys. U septembru 2018. godine, štene staro četiri meseca, sa znacima groznice i petehijalnim krvarenjem, je došlo u ambulantu Klinike za male životinje Fakulteta veterinarske medicine Univerziteta u Beogradu. Hematološka analiza je pokazala trombocitopeniju i blagu anemiju. Pregledom krvnog razmaza ustanovljene su inkluzije u trombocitima. Komercijalni serološki test je pokazao prisustvo antitela protiv Anaplasma spp. Sprovedena je terapija doksiciklinom u trajanju od 14 dana, a posle 48 sati od početka terapije znaci bolesti su se povukli. PCR analizom i senkvencioniranjem je potvrđeno prisustvo Anaplasma platys.

The aim of this study was to investigate the influence of marination, on the growth of Salmonella spp. in contaminated broiler breast fillets during storage. In the conducted study, broiler breast fillets were inoculated with a cocktail of different Salmonella strains and afterwards marinated in different solutions of table salt, sodium tripolyphosphate and/or sodium citrate. The total count of Salmonella spp. was examined on the 0, 3rd, 6th and 9th day of storage. Broiler breast fillets salted in 6% solution of table salt were used as the control. Proximate composition and salt content, pH value and aw value, were determined as the meat quality parameters and parameters which can affect environmental conditions for bacterial growth, as well. Compared to initial contamination, Salmonella spp. count in marinated and salted fillets did not change significantly (prd and 6th day of storage, respectively, but it increased significantly on the 9th day of storage. Marination of broiler breast fillets in different solutions of table salt, sodium tripolyphosphate and/or sodium citrate had a significant influence(pw value, moisture, ash and salt content in marinated broiler meat, but there was no significant influence (p>0.05) on protein and fat content in broiler meat. According to the results obtained it can be concluded that marination of broiler breast fillets in solutions containing table salt, sodium trpolyphosphate and/or citrate, in some way, can prolongate the lag phase of Salmonella spp. growth, where sodium citrate is more effective than sodium tripolyphosphate.

The use of cotton in wound dressing applications is discussed in this chapter. Cotton gauze provides flexibility, controlled adherence to the surrounding tissue, gas permeability, durability and absorbance of fluids exuded from the wound but doesn't provide therapeutic treatment. Therapeutic effect can be provided by incorporation of an active ingredient into gauze. In this chapter, supercritical solvent impregnation of cotton gauze with carvacrol is proposed with an aim to impart antibacterial properties to cotton fibres. State of the art in fabrication of textiles with natural bioactive substances, antibacterial activity of carvacrol loaded materials as well as in the use of supercritical fluids in textile material impregnation is presented. The importance of the application of natural substances with strong antibacterial properties in modern age when the world is facing problem of bacterial resistance to antibiotics is stressed and the future application of cotton as a wound dressing is discussed.

Resistance to methicillin in staphylococci is considered to be one of the most dangerous forms of bacterial resistances to antibiotics. Methicillinresistant staphylococci (MRS) are zoonotic agents which cause local and systemic infections in humans and animals, often with a fatal outcome due to the absence of adequate antibiotic therapy. People colonized with strains of MRS are asymptomatic carriers and reservoirs of these strains in human populations. The aim of this research was to determine the prevalence of strains of MRS among clinically healthy students of the Faculty of Veterinary Medicine in Belgrade. The study was conducted on 100 volunteers: 62 males and 38 females. Given that staphylococci are expected to be found in the highest percentage in the nose and on the armpit skin, the swabs were taken from these regions of each person. Blood agar was innoculated immediately on taking the swabs After the incubation and isolation, the staphylococci were identified to species level. Their susceptibility to methicillin was tested in a disk-diffusion test with cefoxitin. All strains which were found to be resistant to cefoxitin were investigated for the presence of mecA gene with PCR. Staphylococci were isolated in 146 out of the 200 swabs taken: there were 79 nose swabs and 67 axillar swabs positive for these bacteria. Seventeen isolates were resistant to cefoxitin and the presence of the mecA gene was confirmed in seven, four of which were taken from the nose and three from the axillary region. The results of this research show that, being 6%, the prevalence of mecA-positive staphylococci in the population of clinically healthy students of veterinary medicine is significant. The percentage of methicillin-resistant staphylococci was higher in nose than in the axillar region of the students. Rezistencija na meticilin kod stafilokoka smatra se jednim od najopasnijih oblika rezistencije bakterija na antibiotike. Sojevi meticilin rezistentnih stafilokoka (MRS) pripadaju zoonotskim agensima i uzročnici su lokalnih i sistemskih infekcija kod ljudi i životinja, često sa fatalnim ishodom zbog nedostatka efikasne terapije. Ljudi kolonizovani sojevima MRS su asimptomatski nosioci i predstavljaju rezervoare ovih sojeva u humanoj populaciji. Cilj ovog istraživanja je bio da se utvrdi prevalencija izolata MRS kod klinički zdravih studenata Fakulteta veterinarske medicine u Beogradu. U istraživanje je bilo uključeno 100 studenata - dobrovoljaca, 62 muškog i 38 ženskog pola. Brisevi su uzimani iz nosa i pazuha zato što su stafilokoke u najvećem procentu zastupljene u tim regijama. Brisevi su odmah zasejavani na krvni agar. Nakon inkubacije i izolacije, izvršena je identifikacija stafilokoka do vrste. Ispitivanje osetljivosti stafilokoka na meticilin izvedeno je primenom disk difuzione metode sa cefoksitinom. Svi sojevi rezistentni na cefoksitin ispitani su na prisustvo mecA gena metodom lančane reakcije polimeraze (PCR). Od ukupno 200 uzetih briseva izolovano je 146 izolata stafilokoka, 79 iz briseva nosa i 67 iz briseva pazušne regije. Kod 17 izolata ustanovljena je rezistencija na cefoksitin, a kod 7 je utvrđeno prisustvo mecA gena. Četiri mecA pozitivna izolata su poticala iz briseva nosa, a 3 sa kože pazušne regije. Utvrđena prevalencija meticilin-rezistentnih stafilokoka kod klinički zdravih studenata Fakulteta veterinarske medicine od 6%, procenjena je kao značajna. Veća učestalost stafilokoka rezistentnih na meticilin ustanovljena je na sluzokoži nosa u odnosu na kožu pazuha.

Delo obravnava izračun porazdelitve napetosti v navitjih energetskega transformatorja pri preizkusu z udarnim valom in izdelavo pripadajočega programskega orodja. Model je obravnavan s t. i. koncentriranimi parametri, s katerimi tvorimo ustrezno R, L in C vezje za posamezen tip navitja. Opisani so osnovni principi za računanje elementov nadomestnega vezja navitja in izpeljava diferencialnih enačb. Napetosti in tokovi so izračunani z numeričnim reševanjem sistema navadnih diferencialnih enačb. Naloga zajema tudi meritve na realnem transformatorju in preverjanje ustreznosti modela. Programsko orodje je izdelano v programskem jeziku C# z razvojnim orodjem Visual Studio podjetja Microsoft® in uporabniku omogoča pregled rezultatov ter obdelavo teh z ostalimi orodji, kot je na primer Excel. Thesis deals with development of software application for calculation of surge wave distribution over the power transformer winding. Winding is represented with lumped parameter model, which consists of basic linear parameters as resistance, inductance and capacitance. Calculation principles of these elements and derivation of system of differential equations are shown. Voltages and currents of corresponding equivalent circuit are calculated using numerical methods for calculation of ordinary differential equations. Measurements on real power transformer are included in this work as model verification. Program is developed in C# computer language which is part of Microsoft's® Visual Studio. It allows users to overview calculated data and exporting the results for further use in Excel.

Elecampane (Inula helenium L., Asteraceae), a well-known traditional medicinal herb, and its root (Inulae radix) have been used as antiseptics, expectorants, diuretics and bactericidal agents. The aim of this study was to investigate the antimicrobial activity of different Inulae radix isolates against microorganisms that commonly cause skin infections. The essential oil obtained by hydrodistillation (IH-HD) and Sohxlet extracts, ether (IH-SOX-E) and ethanol (IH-SOX-Et) fractions were investigate The isolates were dissolved in DMSO at the concentration of 2560 to 0.625 μg/mL. The antimicrobial activity was tested by using the microdilution method against reference bacterial strains and clinical isolates from skin swabs. IH-SOX-E exhibited a strong antimicrobial activity against all tested strains, IH-HD as well, with the exception of S. aureus, ATCC 25923 (moderate activity), while IH-SOX-Et showed a strong activity only against clinical isolate of C. albicans. The results of our study indicate that elecampane root isolates may be used for incorporation into phytopreparations for topical application in treating skin infections, especially those caused by C. Albicans the most possible sites for interactions with other compounds. Oman (Inula helenium L., Asteraceae) je cenjena lekovita biljka, čiji je koren poznat po antiseptičnom ekspektorantnom, diuretičnom i baktericidnom delovanju. Cilj rada bio je ispitivanje antimikrobne aktivnosti različitih izolata korena omana, Inulae radix protiv mikroorganizama koje uobičajeno izazivaju infekcije kože. Ispitivani su etarsko ulje dobijeno hidrodestilacijom (IH-HD) i Sohxlet ekstrakti i to etarska (IH-SOX-E) i etanolna (IH-SOX-Et) frakcija. Izolati su rastvoreni u DMSO u koncentraciji od 2560 do 0,625 μg/mL. Antimikrobna aktivnost ispitivana je mikrodilucionom metodom u bujonu na referentnim bakterijskim sojevima i kliničkim izolatima iz briseva kože. IH-SOX-E ispoljila je jaku antimikrobnu aktivnost prema svim testiranim mikroorganizmima, IH-HD takođe, osim prema Streptococcus aureus ATCC 25923 (umerena aktivnost), a IH-SOX-Et jaku aktivnost pokazala je jedino prema kliničkom izolatu Candida albicans. Rezultati naših ispitivanja pokazuju da se izolati korena omana mogu koristiti za inkorporiranje u fitopreparate namenjene za spoljašnju primenu kod kožnih infekcija, naročito gljivičnih infekcija izazvanih C. albicans.

Over the past several decades, the medical community has become increasingly concerned over the ability of bacteria to develop resistance to antibacterial drugs. Many bacteria have become resistant to multiple classes of antibiotics, especially in hospitals. Determination of antibiotic resistance mechanisms of pathogenic bacteria can provide information that will help in infection control and surveillance of bacterial resistance to antibiotics. In this study we demonstrated novel, rapid and accurate method for determination of antibiotic resistance mechanisms of pathogenic bacteria. The method is based on proteome analysis exclusively driven by peptide/protein de novo sequencing. The method is based on chemically activated fragmentation in negative and positive ion mode (CAF-/CAF+), a newly developed technique that enables tandem mass spectrometry acquisition (MS/MS) from both positive and negative ion modes. Peptides identified as overlapped amino acid sequences read from both ion modes could unambiguously assign peptide after de novo sequencing and protein database identification. Using this method, the key proteins responsible for the antibiotic resistance of pathogenic bacteria such as clinical Pseudomonas aeruginosa aeruginosa, uropathogenic Escherichia coli and Proteus mirabilis mirabilis were identified. Induction of AmpC enzymes was found in three P. aeruginosa strains resistant to meropenem. Sensitivity of CAF-/CAF+ method enabled even determination of particular mutations between the AmpC enzymes in tested strains. The interaction of various proteins such as β-lactamase and outer membrane protein A was found as mechanism of penicillin G resistance in E. coli MFBF10519. Aforementioned method enabled the determination of differentially expressed proteins (antibiotic susceptibility vs. antibiotic resistance) responsible for specific types of antimicrobial resistivity and is presented as a promising de novo sequencing tool.

In the present study, isolation of essential oils from leaves of rosemary (Rosmarinus officinalis), sage (Salvia officinalis) and thyme (Thymus vulgaris) by supercritical carbon dioxide as well as hydrodistillation was investigated from the point of yield and antimicrobial activity of obtained extracts against Geobacillus stearotermophillus, Bacillus cereus, Bacillus subtilis var. niger, Enterococcus faecium, Salmonella enteritidis and E.coli strains. Thyme isolates had the highest antibacterial efficiency against tested foodborne bacteria strains (MIC=40-640 μg/cm3) followed by rosemary (MIC=320-1280 μg/cm3) and sage (MIC=160-2560 μg/cm3) isolates. Among tested bacteria, Bacillus species were the most sensitive to presence of all tested supercritical extracts and hydrodistillates. . U ovoj studiji je ispitana izolacija etarskih ulja iz lista ruzmarina (Rosmarinus officinalis), žalfije (Salvia officinalis) i timijana (Thymus vulgaris) postupkom natkritične ekstrakcije ugljenik(IV)-oksidom sa aspekta prinosa i antimikrobne aktivnosti dobijenih izolata protiv Geobacillus stearotermophillus, Bacillus cereus, Bacillus subtilis var. niger, Enterococcus faecium, Salmonella enteritidis i E.coli sojeva. Izolati timijana su pokazali najjače antibakterijsko dejstvo u odnosu na testirane sojeve (MIC=40-640 μg/cm3). Zatim slede izolati ruzmarina (MIC=320-1280 μg/cm3) i žalfije (MIC=160-2560 μg/cm3). Među testiranim bakterijama, Bacillus sojevi su se pokazali kao najosetljiviji na prisustvo kako natkritičnih ekstrakata, tako i hidrodestilata. .

Being an important pathogenic and indicator microorganism, E. coli has been included in all the resistance follow up programs worldwide. We carried out sampling on 42 cattle, pig and poultry farms. After sampling the material was delivered to the laboratories, where it was exposed to standard treatment and streaking by standard methods of microbiological diagnostics with the aim to isolate and identify E. coli strains. On the selected isolated E. coli strains an investigation of its sensitivity to a certain number of antimicrobial agents (ampicillin, tetracycline, chloramphenicol streptomycin, neomycin and sulphametoxasol) was carried out. After obtaining the results from each farm for the three different animals categories (young healthy, young unhealthy, adult), 60 E. coli strains for eachcategori were isolated to be examined for sensitivity to 17 antibiotics and chemotherapeutics. The investigation included a total of 180 E. coli strains originating from cattle, pigs and poultry. For the investigation of isolated E. coli strains Mueller Hinton broth and agar, antibiogram tablets (Torlak Belgrade) as well as discs by Becton Dickinson, BBL (USA) were used In isolated E.coli strains resistance to investigated antimicrobial agents was significantly different depending on the species age and health status of the animals. The majority of the isolated E. coli strains examined for 17 antibiotics and chemotherapeutics showed a resistance to 15, and sensitivity only to cephalosporines of the III generation and colistin. U svetu je E.coli kao važan patogeni i indikatorski mikroorganizam uključena u sve programe praćenja rezistencije i upravo iz tih razloga je i u našim uslovima sprovedeno uzorkovanje materijala na 42 farme goveda, svinja i živine. Materijal je po uzorkovanju dopreman do laboratorije nakon čega je vršena obrada i zasejavanje primenom uobičajenih metoda mikrobiološke dijagnostike u cilju izolacije i identifikacije sojeva E.coli. Na odabranim izolovanim sojevima E. coli izvršeno je ispitivanje osetljivosti rezistencije na sledeća antimikrobna sredstva: ampicilin, tetraciklin hloramfenikol, streptomicin, neomicin i sulfametoksazol. Nakon dobijanja rezultata sa svake farme iz tri različite kategorije životinja (mlade zdrave, mlade bolesne, odrasle) izdvojeno je po 60 sojeva E.coli za ispitivanje osetljivosti - rezistencije na 17 antibiotika i hemioterapeutika. Ispitivanjem je obuhvaćeno ukupno 180 sojeva E. coli poreklom od goveda, svinja i živine. U ispitivanju su korišćeni Mueller Hinton agar, antibiogram tablete i diskovi proizvođača Torlak i Becton Dickinson, BBL (USA). Kod izolovanih sojeva E.coli prevalencija rezistencije na ispitivana antimikrobna sredstva značajno se razlikovala u zavisnosti od vrste, starosti i zdravstvenog stanja životinja. Kod većeg broja izolovanih sojeva E.coli ispitanih na 17 antibiotika i hemio­terapeutika ustanovljena je rezistencija na 15 a osetljivost samo na cefalosporine III generacije i kolistin.

Problem of bacteria resistance on antimicrobial agents is not as simple as in the first days of their application. Increased use of antibiotics and chemotherapeutics contributed that a lot of bacteria strains show not only resistance, but also a multiple resistance to a great number, in some cases even to all used antimicrobial agents. Having that in mind a great many of scientific investigations pointed out a continual appearance and spreading of' bacteria resistance sweet as to a justified fear of fatal infectious diseases emerging, without possibility of an efficient antimicrobial therapy. Therefore, the problem becomes very relevant on both local and global levels, especially if it is well known that antibiotics and chemotherapeutics have the effect both on pathogenic and saprophyte bacteria, leading into a growth suppression or elimination of sensitive strains. This characteristic represents both health and ecological problem because of favoring growth of a small number of resistant bacteria strains. Continual and strong selective pressure of antimicrobial agents used in human veterinary medicine, together with genetic abilities of bacteria brings about emergence of greater number of resistant pathogenic bacteria strains. In intensive farming the emergence of greater number of resistant pathogenic bacteria strains is strengthened by antibiotics and chemotherapeutics application in diseases prevention, as well as growth factor, and in healing, but in larger quantities. For these reasons, the prevalence of bacteria resistance in domestic animals, which is in direct correlation with quantity of applied antimicrobial agents, is discussed. This is especially significant in case of domestic animals which products are used in human nutrition. Problem rezistencije bakterija na antimikrobna sredstva nije tako jednostavan kao u pionirskim danima njihove primene. Velika upotreba antibiotika i hemioterapeutika doprinela je da mnogi sojevi bakterija ispoljavaju ne samo rezistenciju već i multipnu rezistenciju na veći broj, a u nekim slučajevima i na sva korišćena antimikrobna sredstva. Imajući to u vidu, brojna naučna istraživanja ukazuju na kontinuirano pojavljivanje i širenje rezistencije kod bakterija i opravdan strah od pojave fatalnih injektivnih oboljenja bez mogućnosti efikasne antimikrobne terapije. Stoga je problem aktuelan ne samo na lokalnom već i na globalnom nivou, posebno ako se zna da antibiotici i hemioterapeutici deluju ne samo na patogene već i na saprofitne bakterije dovodeći do supresije rasta ili eliminisanja osetljivih sojeva. Ova njihova osobina predstavlja ne samo zdravstveni već i ekološki problem zbog favorizovanja rasta malobrojnih rezistentnih sojeva bakterija. Kontinuiran i jak selektivni pritisak antimikrobnih sredstava korišćenih u humanoj veterinarskoj medicini i poljoprivredi, udružen sa genetskim sposobnoslima bakterija uzrokuje pojavu sve većeg broja rezistentnih sojeva patogenih bakterija. U intezivnim uslovima držanja životinja, pojava većeg broja rezistentnih sojeva patogenih bakterija je još vise potencirana primenom antibiotika i hemioterapeutika u preveniranju bolesti, zatim kao faktora rasta i u lečenju, ali u mnogo većim količinama. Iz tih razloga se i govori o prevalenciji rezistencije bakterija kod domaćih životinja koja je u direktnoj korelaciji sa količinom utrošenih antimikrobnih preparata. Ovo posebno ima značaja kada se radi o domaćim životinjama čiji se proizvodi koriste u ishrani ljudi.

Samples (30) of feces of calves, piglets, guilds and pregnant sows with signs of diarrhea, as well as rectal smears, received from farms, where related problems were followed for three months, were used in these investigations. The material was processed so as to isolate microorganisms using the usual microbiological methods (microscopy, sowing cultures on bouillon, Triptose agar gel with 6% sheep's blood, MacConkey agar and Saburaud dextrose agar). After clean cultures were obtained, biochemical identification was performed using commercial ID kits (BBL Crystal Entero/nonfermenter ID kit, Bee-ton Dickinson, USA), as well as standard biochemical series. Resistance, or sensitivity of isolated strains of E.coli, were examined using the disk diffusion method according to Kirby Bauer on Mueller Hinton agar, using antibiogram tablets (Torlak, Belgrade) and antibio-gram discs (Becton Dickinson, USA). Investigations covered'17 antimicrobial preparations ampicillin, amoxicillin with clavulonic acid, tetracycline, chloramphenicol neomycin, streptomycin, gentamycin, trimetoprim, sulphamethoxasole trimetoprin + sufametoxasole, cephalexin, cephachlor, cephtriaxone spectinomycin, cholistin, nalidixin acid, ciprofloxacin. The efficacy and choice of the used antibiotic againstthe isolated strain was performed according to the standard recommendations of the National Committee for Clinical Labora- tory Standards (NCCLS), USA, as well as on the grounds of the instructions of the disc manufacturers. The established prevalence of resistant strains of E.coli to certain antibiotics and chemotherapeutics differed depending on animal age and specie. On the grounds of the analyses of the obtained prevalence of resistant strains in diseased, healthy, young and old animals, it is possible to detect the application of a certain antibiotic in prophylaxis and therapy. At the same time, the obtained results indicate the inadequate and uncontrolled administration of antibiotics to both claves (85%) and piglets (95%), which leads to the high prevalence not only of the resistant, but also of multiresistant strains. U ispitivanjima kao materijal korišćeni su uzorci (30 uzoraka) fecesa teladi, prasadi, nazimica i suprasnih krmača sa znacima dijareje, kao i rektalni brisevi dopremani sa farmi na kojima je problematika praćena tokom tri meseca. Materijal je obrađivan radi izolovanja mikro-organizama uobičajenim mikrobiološkim metodama (mikroskopski, kulturelno-zasejavanjem na hranljivi bujon, Triptozni agar sa 6 % ovčje krvi, MacConkey agar i Saburaud dekstrozni agar). Nakon dobijanja čistih kultura obavljena je biohemijska identifikacija uz primenu komercijalnih ID kitova (BBL Crystal Entero/nonfermenter ID kit, Becton Dickinson, USA), kao i standardnih biohemijskih nizova. Ispitivanje rezistencije, odnosno osetljivosti izolovanih sojeva E. coli obavljeno je disk difuzionom metodom po Kirby Baueru na Mueller Hinton agaru uz korišćenje antibiogram tableta (Torlak Beograd) i antibiogram diskova (Becton Dickinson, USA). Ispitivanjima je bilo obuhvaćeno 17 antimikrobnih sredstava: ampicllin, amoksicilin sa klavulonskom kiselinom, tetraciklin, hloramfenikol, neomicin, streptomicin gentamicin, trimetoprim, sulfametoksazol, trimetoprim + sufametoksazol cefaleksin, cefahlor, ceftriakson, spektinomicin, kolistin, nalidiksinska kiselina i ciprofloksacin. Procena efikasnosti i izbor korišćenog antibiotika u odnosu na izolovani soj izvedeni su prema standardnim preporukama NCCLS (The National Committee for Clinical Laboratory Standards USA), kao i na osnovu uputstva proizvođača diskova. Ustanovljena prevalencija rezistentnih sojeva E. coli prema određenim antibioticima i hemoterapeuticima razlikovala se zavisno od starosti i vrste životinja. Na osnovu analize dobijene prevalencije rezistentnih sojeva kod bolesnih, zdravih, mladih i starijih životinja može da se otkrije primena određenog antibiotika u profilaksi i terapiji. Istovremeno dobijeni rezultatii ukazuju na neadekvatnu i nekontrolisanu primenu antibiotika kako kod teladi (85%) tako i kod prasadi (95%) što dovodi do visoke prevalencije ne samo rezistentnih, već i multirezistentnih sojeva.

A dog aged 12 years was treated by several veterinarians at the Clinic for Surgery, Orthopedics and Ophthalmology of the Faculty for Veterinary Medicine of the University of Belgrade, and the following was diagnozed chronic blepharitis, conjunctivitis, kera-titis, and amblyopia in both eyes. Material for microbiological examinations was taken from both eyes using the method of smears. The smear samples were sown on Triptose agar with 6% sheep's blood, MacConkey agar and Saburaud agar (Torlak). The sown bases were incubated aerobically at 37°C. Characteristic colonies were isolated and the microorganisms were identified using the catalase, oxidase tests, tests for determining the presence of urease enzymes, CAMP phenomenon with Staphylococcus aureus, as well as the rabbit plasma coaggula-tiontest. In addition to standard laboratory methods, BBL Crystal Rapid Gram positive ID system, manufactured by Becton Dickinson, USA, was used for the typization of the isolated type of microorganism. Studying all the above parameters, we obtained absolutely identical results which correspond to the referent values for Rhodococcus equi. Kod psa starog 12 godina, koga je lečilo vise veterinara, na Klinici za hirurgiju, ortopediju i oftalmologiju dijagnostikovani su: hronični blefaritis, konjunktivitis, keratitis, kao i ambliopija oba oka. Materijalza mikrobiološko ispitivanje uzet je iz oba oka metodom brisa. Dostavljeni uzorci briseva zasejavani su na triptozni agar sa 6% ovčije krvi, MacConkey agar i Saburaud agar (Torlak). Zasejane podloge inkubisane su aerobno na temperaturi od 37°C. Posle izolacije karakterističnih kolonija radi identifikacije mikroorganizma korišćeni su testovi katalaze, oksidaze prisustva enzima ureaze, CAMP fenomena sa Staphylococcus aureus, kao i test koagulacije plazme kunića. Pored standardnih laboratorijskih metoda, za tipizaciju izolovane vrste mikroorganizma korišćen je BBL Crystal Rapid Gram positive ID sistem proizvođača Becton Dickinson, USA. Ispitivanjem svih navedenih parametara, dobijeni su potpuno identični rezultati koji odgovaraju referentnim vrednostima za Rhodococcus equi.

Salmonellosis is an important zoonosis manifested in very different clinical picture including enteritis, septicemia, abortion, and combination of disease syndromes. Only 10 to 12 serovars of the Salmonella spp. are capable to causing disease in cattle including Salmonella dublin, S.typhimurium and S. bovismorbificans. In contrast to most other Salmonella serovars, S.dublin shows a high degree of host specificity for cattle. Infection of cattle with S.dublin may be inapparent with carrier state and disease tends to become endemic on certain farms. . Salmoneloza je važna zoonoza sa veoma različitom kliničkom slikom koja manifestuje enteritisom, septikemijom, abortusima i kombinovanim simptomima bolesti. Samo 10 do 12 serovarijeteta Salmonella spp. dovodi do infekcije goveda uključujući Salmonella dublin, S.typhimurium and S. bovismorbificans. .

Streptococcus suis is a normal inhabitant of the respiratory system of pigs. Clinically healthy pigs are a major reservoir of the pathogen, but, as well, the most important link in the epidemiology of infection in humans. Hence, the aim of this study was to establish the presence of S. suis on some pig farms in Serbia and to determine their serotype affiliation. For this research, we tested 226 samples: nasopharyngeal tonsil swabs, nasal cavities swabs, parts of or all nasopharyngeal tonsils, swabs from dead animals, swabs from pig carcasses, and swabs from the butchers knives. Identification of S. suis strains was performed with antisera specific for capsular antigens. S. suis serotype 2 was established in 67.7% cases, and serotypes 7, 9 and 1 were isolated in a much smaller percentage (17.6, 8.8 and 5.9%, respectively). Serotype 2 is the only serotype isolated from all the collected samples. Serotype 9 was isolated from healthy pig tonsil swab and from 2 nasal swabs, and was not found in the samples taken from diseased pigs. The serotype 1 was isolated only from nasopharyngeal tonsil swabs of piglets, while serotype 7 was isolated from nasopharyngeal tonsil swabs, nasopharyngeal tonsil sections, but also from nasal swabs of healthy pigs. It can be concluded that the infections caused by different serotypes of S. suis are present in the herd of pigs in Serbia; hence, it is very important to identify what serotypes of S. suis are involved in pathogenesis of infections in pigs.

U ovoj doktorskoj disertaciji je ispitana mogućnost primene simpleks i multipleks PCR protokola sa prethodno opisanim prajmerima za preciznu identifikaciju najčešće izolovanih serovarijeteta Salmonella vrsta na teritoriji Vojvodine. Takođe je ispitana efikasnost primene metode sekvenciranja gena za 16Ѕ rRNK u preciznoj identifikaciji salmonela. Ispitano je 107 izolata Salmonella enterica ssp. enterica identifikovanih klasičnom serotipizacijom kao: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). Korišćena je tripleks PCR reakcija u detekciji bcfC, steB i sdf lokusa, optimizovana tokom preliminarnih ispitivanja. Primenom tripleks PCR, samo S. Enteritidis je mogla precizno da bude razlikovana od drugih serovarijeteta, dok su ostali serovarijeteti na osnovu rezultata tripleks PCR svrstavani u dve grupe (Grupa 1 i Grupa2). S. Infantis, najzastupljeniji serovarijetet među ispitanim izolatima nije mogao biti precizno identifikovan jer se primenom tripleks PCR nije razlikovao od ostalih članova Grupe 2. Za njegovu konačnu identifikaciju je primenjen simpleks PCR za umnožavanje segmenta fljB koji je karakterističan samo za S. Infantis. Sekvenciranje gena za 16Ѕ rRNK je primenjeno u identifikaciji izolata devet različitih serovarijeteta. Poredjenjem rezultata dobijenih u klasičnoj serotipizaciji i u PCR, uočeno je slaganje u 91 slučaju od ukupno 107 (85%). Od 33 izolata S. Enteritidis njih 31 (94%) je dao očekivan PCR profil. U slučaju S. Infantis PCR je potvrdila identifikaciju u samo 75% (39 od 52) slučajeva. Ostali ispitani serovarijeteti dali su očekivane PCR profile. Sekvenciranje gena za 16Ѕ rRNK je bilo pouzdano samo u određivanju pripadnosti ispitanih izolata rodu Salmonella. Aim of this doctoral thesis was to test the applicability of published primers for the identification of serovars isolated most frequently in the north part of our country using multiplex and simplex PCR reactions. The efficacy of sequencing of gene for 16S ribosomal RNA for identification of Salmonella strains was also checked. The study was conducted on 107 serotyped Salmonella enterica ssp. enterica isolates. Strains belonged to the following serovars: Infantis (52), Enteritidis (33), Tenessee (5), Mbandaka (4), Montevideo (3), Havana (2), Lille (2), Senftenberg (2), Typhimurium (1), Agona (1), Derby (1) i Livingstone (1). A triplex PCR protocol (bcfC, steB, sdf) optimized during preliminary investigations was used. This reaction separated S. Enteritidis from other strains, while the remaining serovars divided in two distinct groups (Group 1 and Group 2). As the used PCR protocol could not differentiate S. Infantis, the most numerous serovar among isolates, from other members of the Group 2, its identification was completed using a simplex PCR reaction targeting fljB specific for S. Infantis. Comparison of the results of serotyping and PCR protocols revealed a concordance in 91 cases from the total of 107 (85%). From 33 strains serotyped as S. Enteritidis 31 (94%) had the predicted PCR profile. Regarding S. Infantis, identification based on genomic markers confirmed this serovar only in 75% of cases (39 from 52). Other serovars showed their typical PCR profiles. Sequencing of gene for 16S ribosomal RNA could reliably determine only that tested isolates are the members of the genus Salmonella

Nosema ceranae je микроспоридијa којa паразитира код Европске медоносне пчеле, Apis mellifera, и доминантна је врста рода Nosema у југоисточној Европи. У зависности од степена инфекције и периода године, овај ендопаразит може имати далекосежне последице по пчеле, њихово здравље и продуктивност. Последњих година посебно је истраживан утицај N. ceranae на имунитет пчела а закључци појединих истраживања издвајају ефекат супресије гена значајних за имунитет пчела, односно доказују имуносупресивне последице инфекције врстом N. ceranae. Терапија ноземозе подразумева употребу антибиотика фумагилина, који има доказану ефикасност у сузбијању инфекције али његови споредни ефекти могу представљати проблем за саме пчеле и квалитет пчелињих производа, те је стога је његова примена у многим земљама ограничена. Имајући ово на уму константно се трага за природним и синтетским компонентама које би биле замена за фумагилин. Тестиране су многе супстанце, а међу онима које су показале најбољи ефекат издвајају се екстракти биљака и алги и синтетске витаминско-минералне мешавине. У овом раду је у кавезном експерименту тестирано пет препарата: (1) антибиотик фумагилин, (2) биљни екстракт тимол; и три адитива у исхрани пчела: (3) комерцијални препарат Beewell AminoPlus који представља витаминско-аминокиселинску мешавину, (4) комерцијални препарат Medenko forte који садржи екстракте храстове коре, пелена и жалфије и (5) полисахаридни екстракт гљиве Agaricus blazei. Пчеле су у свим третираним групама трећег дана живота инфициране спорама N. ceranae, а различите групе су третиране препаратима од првог, трећег и шестог дана. Код третираних пчела је праћен ефекат на преживљавање пчела, степен Nosema инфекције, нивое експресије гена значајних за имунитет пчела (абецин, дефензин, хименоптецин, апидецин и вителогенин) и ниво оксидативног стреса праћен кроз активност антиоксидативних ензима (каталазе, супероксид дизмутазе и глутатион С-трансферазе) и концентрације малондиалдехида... Apis mellifera, and the dominant Nosema species in Southeast Europe. Depending on the infection level it could have significant consequences on bees, their health and productivity. The impact of N. ceranae on honey bees’ immunity has been particularly investigated in recent years. Some conclusions of this research underline N. ceranae-induced suppression of immune-related genes, proving its immunosuppressive impact. Treatment for nosemosis includes the use of the antibiotic fumagillin. Fumagillin has proven effect in Nosema control, but its side effects may pose a problem for the bees and the quality of their products, which is why its application in many countries have been limited. Hence, researchers are constantly looking for natural and synthetic components which could be suitable substitutes for fumagillin. Many products have been tested and plant/algae extracts and synthetic vitamin-mineral complexes showed the best effects. This laboratory (cage) experiment included testing of five products: (1) antibiotic fumagillin (2) a plant extract - thymol, and three dietary supplements: (3) a commercial amino acid and vitamin complex named Beewell AminoPlus, (4) a commercial mixture of oak bark, sage and absinth extracts named Medenko forte, and (5) a polysaccharide extract of Agaricus blazei mushroom. Bees in all treatment groups were infected with N. ceranae spores on day 3, and treated with the listed products from day 1, 3 or 6 after emergence. Bee survival, Nosema loads, levels of immune-related genes (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) expression and the level of oxidative stress, assessed through the activity of antioxidative enzymes (catalase, glutathione S-transferase and superoxide dismutase) and the concentration of malondialdehyde, were measured in all experimental groups. The obtained values were compared with those of control (noninfected and infected) groups...

U ovoj doktorskoj disertaciji ispitano je prisustvo uzročnika kju groznice - bakterije Coxiella burnetii (C. burnetii) kod nevlasničkih pasa poreklom sa teritorije grada Beograda, primenom molekularnih i seroloških metoda. Molekularni metod je korišćen za otkrivanje uzročnika u reproduktivnim tkivima nevlasničkih pasa, kao i u krpeljima prisutnim na ispitivanim životinjama, dok je imunoenzimskim testom utvrđeno prisustvo specifičnog serološkog odgovora kod ispitivanih životinja. Za otkrivanje prisustva DNK C. burnetii u uzrocima korišćena je lančana reakcija polimeraze (Trans-PCR) kojom se umnožava IS1111 fragment C. burnetii, dok je za otkrivanje specifičnih antitela protiv C. burnetii u serumima ispitivanih pasa korišćen modifikovani komercijalni imunoenzimski test (ELISA). U istraživanju je sakupljeno 316 krpelja sa 51 nevlasničkog psa i identifikovane su tri vrste krpelja: Rhipicephalus sanguineus, Ixodes ricinus i Dermacentor reticulatus. Prisustvo DNK C. burnetii ustanovljeno je u 10,53% (24/228) krpelja vrste R. sanguineus, koji su uzorkovani sa sedam pasa. Reproduktivna tkiva pasa sakupljena su od ukupno 105 pasa - 74 ženke (70,48%) i 31 mužjaka (29,52%). DNK C. burentii je ustanovljena kod 20,95% (22/105) pasa i to u 16,13% (5/31) uzoraka semenika pasa, odnosno u 22,97% (17/74) uzoraka materica i jajnika. Kod 29,52% (31/105) pasa je 5 ustanovljeno prisustvo specifičnih antitela protiv C. burnetii i to 32,26% (10/31) mužjaka i 28,38% (21/74) ispitanih ženki... In this doctoral dissertation, the presence of the causative agent of Q fever - the bacterium Coxiella burnetii (C. burnetii) in stray dogs has been examined using molecular and serological methods. Molecular method was used for the detection of the agent in the reproductive tissues of stray dogs, as well as in the ticks recovered from the examined animals, while immunoenzyme test was used for the detection of specific serological response in the examined animals. A polymerase chain reaction (Trans-PCR) targeting IS1111 element of C. burnetii was used for the detection of C. burnetii DNA in the samples, while a modified commercial immunoassay (ELISA) was used for the detection of specific antibodies against C. burnetii in the sera of the examined dogs. In this study, 316 ticks were recovered from 51 stray dogs and three tick species were identified: Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus. The presence of C. burnetii DNA was established in 10.53% (228/24) R. sanguineus ticks, which originated from seven dogs. Reproductive tissues of dogs were collected from a total of 105 dogs - 74 females (70.48%) and 31 males (29.52%). The presence of C. burentii DNA has been detected in 20.95% (22/105) dogs. C. burnetii DNA was detected in 7 16.13% (5/31) samples of dog testicles and in 22.97% (17/74) samples of uteri and ovaries...

In this doctoral dissertation, the presence of the causative agent of Q fever - the bacterium Coxiella burnetii (C. burnetii) in stray dogs has been examined using molecular and serological methods. Molecular method was used for the detection of the agent in the reproductive tissues of stray dogs, as well as in the ticks recovered from the examined animals, while immunoenzyme test was used for the detection of specific serological response in the examined animals. A polymerase chain reaction (Trans-PCR) targeting IS1111 element of C. burnetii was used for the detection of C. burnetii DNA in the samples, while a modified commercial immunoassay (ELISA) was used for the detection of specific antibodies against C. burnetii in the sera of the examined dogs. In this study, 316 ticks were recovered from 51 stray dogs and three tick species were identified: Rhipicephalus sanguineus, Ixodes ricinus and Dermacentor reticulatus. The presence of C. burnetii DNA was established in 10.53% (228/24) R. sanguineus ticks, which originated from seven dogs. Reproductive tissues of dogs were collected from a total of 105 dogs - 74 females (70.48%) and 31 males (29.52%). The presence of C. burentii DNA has been detected in 20.95% (22/105) dogs. C. burnetii DNA was detected in 7 16.13% (5/31) samples of dog testicles and in 22.97% (17/74) samples of uteri and ovaries... U ovoj doktorskoj disertaciji ispitano je prisustvo uzročnika kju groznice - bakterije Coxiella burnetii (C. burnetii) kod nevlasničkih pasa poreklom sa teritorije grada Beograda, primenom molekularnih i seroloških metoda. Molekularni metod je korišćen za otkrivanje uzročnika u reproduktivnim tkivima nevlasničkih pasa, kao i u krpeljima prisutnim na ispitivanim životinjama, dok je imunoenzimskim testom utvrđeno prisustvo specifičnog serološkog odgovora kod ispitivanih životinja. Za otkrivanje prisustva DNK C. burnetii u uzrocima korišćena je lančana reakcija polimeraze (Trans-PCR) kojom se umnožava IS1111 fragment C. burnetii, dok je za otkrivanje specifičnih antitela protiv C. burnetii u serumima ispitivanih pasa korišćen modifikovani komercijalni imunoenzimski test (ELISA). U istraživanju je sakupljeno 316 krpelja sa 51 nevlasničkog psa i identifikovane su tri vrste krpelja: Rhipicephalus sanguineus, Ixodes ricinus i Dermacentor reticulatus. Prisustvo DNK C. burnetii ustanovljeno je u 10,53% (24/228) krpelja vrste R. sanguineus, koji su uzorkovani sa sedam pasa. Reproduktivna tkiva pasa sakupljena su od ukupno 105 pasa - 74 ženke (70,48%) i 31 mužjaka (29,52%). DNK C. burentii je ustanovljena kod 20,95% (22/105) pasa i to u 16,13% (5/31) uzoraka semenika pasa, odnosno u 22,97% (17/74) uzoraka materica i jajnika. Kod 29,52% (31/105) pasa je 5 ustanovljeno prisustvo specifičnih antitela protiv C. burnetii i to 32,26% (10/31) mužjaka i 28,38% (21/74) ispitanih ženki...

Nosema ceranae is a microsporidian endoparasite of the European honey bee, Apis mellifera, and the dominant Nosema species in Southeast Europe. Depending on the infection level it could have significant consequences on bees, their health and productivity. The impact of N. ceranae on honey bees’ immunity has been particularly investigated in recent years. Some conclusions of this research underline N. ceranae-induced suppression of immune-related genes, proving its immunosuppressive impact. Treatment for nosemosis includes the use of the antibiotic fumagillin. Fumagillin has proven effect in Nosema control, but its side effects may pose a problem for the bees and the quality of their products, which is why its application in many countries have been limited. Hence, researchers are constantly looking for natural and synthetic components which could be suitable substitutes for fumagillin. Many products have been tested and plant/algae extracts and synthetic vitamin-mineral complexes showed the best effects. This laboratory (cage) experiment included testing of five products: (1) antibiotic fumagillin (2) a plant extract - thymol, and three dietary supplements: (3) a commercial amino acid and vitamin complex named Beewell AminoPlus, (4) a commercial mixture of oak bark, sage and absinth extracts named Medenko forte, and (5) a polysaccharide extract of Agaricus blazei mushroom. Bees in all treatment groups were infected with N. ceranae spores on day 3, and treated with the listed products from day 1, 3 or 6 after emergence. Bee survival, Nosema loads, levels of immune-related genes (abaecin, apidaecin, hymenoptaecin, defensin and vitellogenin) expression and the level of oxidative stress, assessed through the activity of antioxidative enzymes (catalase, glutathione S-transferase and superoxide dismutase) and the concentration of malondialdehyde, were measured in all experimental groups. The obtained values were compared with those of control (noninfected and infected) groups... Nosema ceranae je mikrosporidija koja parazitira kod Evropske medonosne pčele, Apis mellifera, i dominantna je vrsta roda Nosema u jugoistočnoj Evropi. U zavisnosti od stepena infekcije i perioda godine, ovaj endoparazit može imati dalekosežne posledice po pčele, njihovo zdravlje i produktivnost. Poslednjih godina posebno je istraživan uticaj N. ceranae na imunitet pčela a zaključci pojedinih istraživanja izdvajaju efekat supresije gena značajnih za imunitet pčela, odnosno dokazuju imunosupresivne posledice infekcije vrstom N. ceranae. Terapija nozemoze podrazumeva upotrebu antibiotika fumagilina, koji ima dokazanu efikasnost u suzbijanju infekcije ali njegovi sporedni efekti mogu predstavljati problem za same pčele i kvalitet pčelinjih proizvoda, te je stoga je njegova primena u mnogim zemljama ograničena. Imajući ovo na umu konstantno se traga za prirodnim i sintetskim komponentama koje bi bile zamena za fumagilin. Testirane su mnoge supstance, a među onima koje su pokazale najbolji efekat izdvajaju se ekstrakti biljaka i algi i sintetske vitaminsko-mineralne mešavine. U ovom radu je u kaveznom eksperimentu testirano pet preparata: (1) antibiotik fumagilin, (2) biljni ekstrakt timol; i tri aditiva u ishrani pčela: (3) komercijalni preparat Beewell AminoPlus koji predstavlja vitaminsko-aminokiselinsku mešavinu, (4) komercijalni preparat Medenko forte koji sadrži ekstrakte hrastove kore, pelena i žalfije i (5) polisaharidni ekstrakt gljive Agaricus blazei. Pčele su u svim tretiranim grupama trećeg dana života inficirane sporama N. ceranae, a različite grupe su tretirane preparatima od prvog, trećeg i šestog dana. Kod tretiranih pčela je praćen efekat na preživljavanje pčela, stepen Nosema infekcije, nivoe ekspresije gena značajnih za imunitet pčela (abecin, defenzin, himenoptecin, apidecin i vitelogenin) i nivo oksidativnog stresa praćen kroz aktivnost antioksidativnih enzima (katalaze, superoksid dizmutaze i glutation S-transferaze) i koncentracije malondialdehida...

Pseudomonas aeruginosa je ubikvitaran, oportunistički patogen, uzročnik infekcija kod životinja i ljudi, koje su najčešće povezane sa kompromitovanim imunološkim sistemom domaćina. Patogeneza pseudomonasne infekcije je multifaktorijalan proces koji uključuje produkciju faktora virulencije vezanih za ćeliju, odnosno faktora virulencije koji se sekretuju, složenu međubakterijsku komunikaciju putem četiri sistema, kao i sposobnost prelaska iz planktonske životne forme u formu biofilma. Značaj ove vrste za medicinu i veterinu potvrdila je Svetska zdravstvena organizacija u izveštaju objavljenom 2017. godine, po kom P. aeruginosa pripada kritičnoj grupi bakterijskih vrsta za koje je potrebno hitno razviti nove terapeutike jer, zbog sve učestalije pojave multirezistentnih sojeva, ova vrsta predstavlja ozbiljnu pretnju javnom zdravlju. U okviru ove disertacije uporedno su analizirani faktori virulencije izolata P. aeruginosa izolovanih iz uzoraka obolelih životinja i ljudi, kao što su sposobnost formiranja biofilma (ukupan biofilm i biofilm na granici vazduh-tečnost), proizvodnja piocijanina, hemolitička aktivnost, sposobnost manifestovanja tri tipa pokretljivosti (plivanje, rojenje i grčenje), kao i citotoksični efekat na ćelijama iz ćelijske linije A549. Filogenetska srodnost i grupisanje odabranih izolata P. aeruginosa proučavana je na osnovu parcijalne sekvence gena za 16S rRNK. Ispitivanje postojanja korelacija između odabranih faktora virulencije urađeno je primenom robusne rang korelacije. Grupisanje izolata je izvedeno metodom hijerarhijske klasterizacije na osnovu euklidske udaljenosti normalizovane vrednostima sedam ispitivanih faktora virulencije i citotoksičnosti. Metodom mašinskog učenja konstruisan je prediktivni model na osnovu koga je određena značajnost ispitivanih faktora virulencije u predviđanju citotoksičnog potencijala izolata P. aeruginosa. Ispitivanje razlika između faktora virulencije izolata različitog porekla (izolati poreklom od ljudi u odnosu na izolate poreklom od životinja, odnosno izolati poreklom iz uzorka tečnosti u odnosu na izolate poreklom iz briseva) je izvedeno Wilcoxon Rank-Sum testom i Relative effect testom...

Šiga toksin produkujuće Escherichia coli (STEC) čine jednu od šest grupa dijarejagenih E. coli. Na svjetskom nivou, infekcija izazvana STEC najčešći je uzrok akutne renalne insuficijencije kod djece i starijih osoba. Kako su psi prepoznati kao rezervoari STEC, blizak kontakt ljudi i pasa predstavlja rizik za zoonotsku transmisiju ovih bakterija. U Republici Srbiji, osim ograničenog broja istraživanja o prisustvu STEC kod domaćih životinja i ljudi nema dostupnih literaturnih podataka o istraživanjima ove grupe E. coli kod pasa. Stoga, cilj ove doktorske disertacije bio je da se dokaže prisustvo i utvrdi prevalencija STEC u populaciji pasa s teritorije grada Novog Sada, izvrši tipizacija i molekulrna karakterizacija sojeva E. coli izolovanih iz fecesa pasa i utvrdi prisustvo rezistentnih sojeva E. coli. U ovo istraživanje bio je uključen 101 pas s teritorije Novog Sada, a kao materijal korišćeni su uzorci fecesa pasa. U cilju izolacije E. coli iz fecesa pasa primijenjeni su standardni bakteriološki metodi izolacije i biohemijske identifikacije, a potvrda pripadnosti vrsti izvršena je korišćenjem matricom potpomognute laserske desorpcije/jonizacije-vrijeme preleta masene spektrometrije (MALDI-TOF MS). Za utvrđivanje prisustva STEC kod pasa primijenjeni su metodi lateks aglutinacije za serogrupu O157, direktne aglutinacije za serogrupe “velike šestorke” O26, O45, O103, O111, O121 i O145, test verocitotoksičnosti (VCA), imunoenzimski test (ELISA), kao i lančana reakcija polimeraze (PCR). Antibiotska rezistencija utvrđena je disk difuzionim metodom prema standardima EUCAST i CLSI. U ovom istraživanju, po prvi put u Republici Srbiji, dokazano je prisustvo STEC u populaciji pasa. Primjenom VCA metoda ustanovljena je prevalencija od 1,98%, dok je primjenom ELISA i PCR metoda ustanovljena prevalencija od 5,94% odnosno 4,95%. Dva izolata E. coli pripadala su nekoj od serogrupa “velike šestorke” STEC, dok prisustvo sojeva iz serogrupe O157 nije utvrđeno. U ispitivanoj populaciji pasa utvrđeno je prisustvo sojeva E. coli rezistentnih na jedan ili više korišćenih antibiotika, kao i jednog multirezistentnog izolata. Najviše izolata E. coli pokazalo je rezistenciju na ampicilin (22,5%), dok nijedan izolat nije pokazao rezistenciju na gentamcin. Pozitivna povezanost utvrđena je između stila života psa i prisustva STEC. Naime, značajno veća prevalencija STEC, u odnosu na vlasničke pse, zabilježena je kod pasa lutalica, što je vjerovatno posljedica neograničene slobode kretanja, a time i češćeg kontakta s kontaminiranom hranom i vodom kao izvorom STEC. Nalaz STEC i izolata E. coli rezistentnih na antibiotike kod pasa od značaja je kako s aspekta male kliničke prakse tako i s aspekta javnog zdravlja u Republici Srbiji. Datum Shiga toxin-producing Escherichia coli (STEC) strains constitute one of six groups of diarrheagenic E. coli. Infection caused by STEC is the most common cause of acute renal failure in young children and elderly people worldwide. As dogs are recognized as a reservoir for STEC, the close contact between humans and dogs poses a risk for zoonotic transmission of these bacteria. Except scarce studies of STEC in humans and domestic animals, there are no available data about this E. coli group in dogs in the Republic of Serbia. Therefore the aim of this doctoral dissertation was to determine the presence and prevalence of STEC in the dog populaton in the area of Novi Sad, to perform typing and molecular characterization of E. coli strains isolated from faeces of dogs, as well to determine the presence of antibiotic resistant E. coli strains. In total 101 dogs from the area of Novi Sad was included in this research. As the material fecal samples of dogs were used. In order to isolate E. coli strains from faeces standard methods of bacterial isolation and biochemical identification were used, and for the confirmation of species identity matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was used. In order to determine the presence of STEC in dogs latex agglutination for the serogroup O157, direct agglutination for “big six” serogroups (O26, O45, O103, O111, O121 and O145), Vero cell cytotoxicity assay (VCA), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were used. Antibiotic resistance was determined by disc diffusion method according to the EUCAST and CLSI guidelines. In this research, for the first time in the Republic of Serbia, the presence of STEC has been proven in the dog population. By VCA method the prevalence of 1,98% was determined, while by ELISA and PCR the determined prevalence was 5,94% and 4,95% respectively. Two E. coli isolates belonged to some of the STEC “big six” serogroups, while none of the isolates belonged to O157 serogroup. Furthermore, in the dog population included in this research, the presence of strains resistant to one or more of used antibiotics was determined, as well as the presence of one multiresistant strain. Most of the E coli isolates showed resistance to ampicillin (22,5%), while every of the isolates showed susceptibility to gentamicin. In this research significant association between lifestyle of dogs and STEC harbouring. That is to say, significantly higher STEC prevalence was noticed in stray dogs in comparison to pet dogs, what is likely due to unlimited freedom of movement and thereby more frequent contact with contaminated water and food as sources of STEC. Finding of STEC and E. coli strains resistant to antibiotics in dog population is of importance not just in small animal practice, but also in terms of public health in the Republic of Serbia.

Šiga toksin produkujuće Escherichia coli (STEC) čine jednu od šest grupa dijarejagenih E. coli. Na svjetskom nivou, infekcija izazvana STEC najčešći je uzrok akutne renalne insuficijencije kod djece i starijih osoba. Kako su psi prepoznati kao rezervoari STEC, blizak kontakt ljudi i pasa predstavlja rizik za zoonotsku transmisiju ovih bakterija. U Republici Srbiji, osim ograničenog broja istraživanja o prisustvu STEC kod domaćih životinja i ljudi nema dostupnih literaturnih podataka o istraživanjima ove grupe E. coli kod pasa. Stoga, cilj ove doktorske disertacije bio je da se dokaže prisustvo i utvrdi prevalencija STEC u populaciji pasa s teritorije grada Novog Sada, izvrši tipizacija i molekulrna karakterizacija sojeva E. coli izolovanih iz fecesa pasa i utvrdi prisustvo rezistentnih sojeva E. coli. U ovo istraživanje bio je uključen 101 pas s teritorije Novog Sada, a kao materijal korišćeni su uzorci fecesa pasa. U cilju izolacije E. coli iz fecesa pasa primijenjeni su standardni bakteriološki metodi izolacije i biohemijske identifikacije, a potvrda pripadnosti vrsti izvršena je korišćenjem matricom potpomognute laserske desorpcije/jonizacije-vrijeme preleta masene spektrometrije (MALDI-TOF MS). Za utvrđivanje prisustva STEC kod pasa primijenjeni su metodi lateks aglutinacije za serogrupu O157, direktne aglutinacije za serogrupe “velike šestorke” O26, O45, O103, O111, O121 i O145, test verocitotoksičnosti (VCA), imunoenzimski test (ELISA), kao i lančana reakcija polimeraze (PCR). Antibiotska rezistencija utvrđena je disk difuzionim metodom prema standardima EUCAST i CLSI. U ovom istraživanju, po prvi put u Republici Srbiji, dokazano je prisustvo STEC u populaciji pasa. Primjenom VCA metoda ustanovljena je prevalencija od 1,98%, dok je primjenom ELISA i PCR metoda ustanovljena prevalencija od 5,94% odnosno 4,95%. Dva izolata E. coli pripadala su nekoj od serogrupa “velike šestorke” STEC, dok prisustvo sojeva iz serogrupe O157 nije utvrđeno. U ispitivanoj populaciji pasa utvrđeno je prisustvo sojeva E. coli rezistentnih na jedan ili više korišćenih antibiotika, kao i jednog multirezistentnog izolata. Najviše izolata E. coli pokazalo je rezistenciju na ampicilin (22,5%), dok nijedan izolat nije pokazao rezistenciju na gentamcin. Pozitivnapovezanost utvrđena je između stila života psa i prisustva STEC. Naime, značajno veća prevalencija STEC, u odnosu na vlasničke pse, zabilježena je kod pasa lutalica, što je vjerovatno posljedica neograničene slobode kretanja, a time i češćeg kontakta s kontaminiranom hranom i vodom kao izvorom STEC. Nalaz STEC i izolata E. coli rezistentnih na antibiotike kod pasa od značaja je kako s aspekta male kliničke prakse tako i s aspekta javnog zdravlja u Republici Srbiji.Datum, Shiga toxin-producing Escherichia coli (STEC) strains constitute one of six groups of diarrheagenic E. coli. Infection caused by STEC is the most common cause of acute renal failure in young children and elderly people worldwide. As dogs are recognized as a reservoir for STEC, the close contact between humans and dogs poses a risk for zoonotic transmission of these bacteria. Except scarce studies of STEC in humans and domestic animals, there are no available data about this E. coli group in dogs in the Republic of Serbia. Therefore the aim of this doctoral dissertation was to determine the presence and prevalence of STEC in the dog populaton in the area of Novi Sad, to perform typing and molecular characterization of E. coli strains isolated from faeces of dogs, as well to determine the presence of antibiotic resistant E. coli strains. In total 101 dogs from the area of Novi Sad was included in this research. As the material fecal samples of dogs were used. In order to isolate E. coli strains from faeces standard methods of bacterial isolation and biochemical identification were used, and for the confirmation of species identity matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was used. In order to determine the presence of STEC in dogs latex agglutination for the serogroup O157, direct agglutination for “big six” serogroups (O26, O45, O103, O111, O121 and O145), Vero cell cytotoxicity assay (VCA), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) were used. Antibiotic resistance was determined by disc diffusion method according to the EUCAST and CLSI guidelines. In this research, for the first time in the Republic of Serbia, the presence of STEC has been proven in the dog population. By VCA method the prevalence of 1,98% was determined, while by ELISA and PCR the determined prevalence was 5,94% and 4,95% respectively. Two E. coli isolates belonged to some of the STEC “big six” serogroups, while none of the isolates belonged to O157 serogroup. Furthermore, in the dog population included in this research, the presence of strains resistant to one or more of used antibiotics was determined, as well as the presence of one multiresistant strain.Most of the E coli isolates showed resistance to ampicillin (22,5%), while every of the isolates showed susceptibility to gentamicin. In this research significant association between lifestyle of dogs and STEC harbouring. That is to say, significantly higher STEC prevalence was noticed in stray dogs in comparison to pet dogs, what is likely due to unlimited freedom of movement and thereby more frequent contact with contaminated water and food as sources of STEC. Finding of STEC and E. coli strains resistant to antibiotics in dog population is of importance not just in small animal practice, but also in terms of public health in the Republic of Serbia.

Bruceloza pasa je bolest poznata preko četiri decenije, ali i pored toga ne postoje standardizovani dijagnostički protokoli, kao ni generalni dogovor o najprikladnijem dijagnostičkom testu. Svaka laboratorija definiše sopstvene kriterijume. Ovakva raznovrsnost testova i nedostatak jasno definisanih protokola dovodi do teškoća u interpretaciji rezultata seroloških testova u različitim laboratorijama. Iz tog razloga, cilj ove doktorske disertacije obuhvatio je unapređenje dijagnostike primenom preporučenih i novih, sopstveno pripremljenih testova. Jedan od zadataka ove disertacije je bilo i ispitivanje upotrebljivosti Bruce-ladder multiplex PCR metode za ispitivanje kliničkih uzoraka odnosno tkiva uterusa i testisa pasa. Sakupljen je materijal (krv, testisi i materice) od 225 nevlasničkih pasa i to 145 ženki i 80 mužjaka. Dobijeni rezultati su pokazali da je od ukupno 225 ispitanih uzoraka, 33 ili 14,67% krvnih seruma imalo merljiv titar antitela u 2-ME TAT. Najniži ispitivani titar od 1/50 imalo je 13 krvnih seruma ili 5,78%, kod 8 krvnih seruma ustanovljen je titar od 1/100 ili 3,55%, dok je titar od 1/200 imalo 12 uzoraka seruma ili 5,33%. Primenom klasičnih bakterioloških metoda, B. canis je izolovana iz tri uzorka homogenizata tkiva reproduktivnih organa (1,33%) i to iz 2 uzorka poreklom od mužjaka i jednog uzorka poreklom od ženke. Jedan izolat je poticao od serološki negativnog psa. Od 225 uzoraka ispitanih Bruce-ladder multiplex PCR metodom, pozitivna reakcija je ustanovljena kod dva (0,88%). Pre formulacije indirektnog ELISA testa, određivane su koncentracije proteina. Elektroforetska analiza antigena dobijenih toplotom i ultrazvukom kao i denzitometrijska kvantifikacija su pokazale da je u antigenu dobijenom toplotom, najzastupljenija frakcija molekulske mase 10,95 kDa sa učešćem od čak 43,12% koja odgovara R-LPS-u brucela. Ista frakcija je u antigenu dobijenom ultrazvukom bila zastupljena sa 11,56%, odnosno u količini koja je bila 3,7x manja... The scientific community has known about canine brucellosis for over four decades, even so, there are no standardized diagnostic protocols, nor a general agreement on the most appropriate diagnostic test. Each laboratory defines its own criteria. This variety of tests and the lack of clearly defined protocols lead to difficulties in interpreting the results of serological tests in different laboratories. For this reason, the goal of this doctoral dissertation has been to improve diagnostics using recommended and new, self-prepared tests. One of the tasks of this dissertation was to examine the usability of the Bruce-ladder multiplex PCR method for testing clinical samples, i.e. dog uterine and testicular tissues. The material (blood, testicles and uteruses) was collected from 225 dogs without owners, 145 female and 80 male dogs. The results obtained showed that from a total of 225 tested samples, 33 or 14,67% of blood sera had measurable antibody titer in 2-ME TAT. 13 or 5,78% blood sera had the lowest tested titer of 1/50, in 8 or 3,55% blood sera a titer of 1/100 was determined, while 12 serum samples or 5,33% had the titer of 1/200. By applying classic bacteriological methods, B. canis was isolated from three samples of homogenized reproductive organ tissue (1,33%), 2 from samples originating from males and one specimen originating from a female. One isolate originated from a serologically negative dog. Of the 225 samples assayed using the Bruce-ladder multiplex PCR method, a positive reaction was established in two (0,88%). Protein concentrations were determined prior to the formulation of the indirect ELISA test. The electrophoretic analysis of antigens retrieved using heat and ultrasound, as well as the densitometric quantification, showed that the antigen retrieved using heat had the most prevalent molecular weight fraction of 10,95 kDa with a participation of as much as 43,12% that corresponds to Brucella R LPS. The same fraction was represented in the antigen retrieved using ultrasound with 11,56%, or in a quantity that was 3,7 times lower...

The scientific community has known about canine brucellosis for over four decades, even so, there are no standardized diagnostic protocols, nor a general agreement on the most appropriate diagnostic test. Each laboratory defines its own criteria. This variety of tests and the lack of clearly defined protocols lead to difficulties in interpreting the results of serological tests in different laboratories. For this reason, the goal of this doctoral dissertation has been to improve diagnostics using recommended and new, self-prepared tests. One of the tasks of this dissertation was to examine the usability of the Bruce-ladder multiplex PCR method for testing clinical samples, i.e. dog uterine and testicular tissues. The material (blood, testicles and uteruses) was collected from 225 dogs without owners, 145 female and 80 male dogs. The results obtained showed that from a total of 225 tested samples, 33 or 14,67% of blood sera had measurable antibody titer in 2-ME TAT. 13 or 5,78% blood sera had the lowest tested titer of 1/50, in 8 or 3,55% blood sera a titer of 1/100 was determined, while 12 serum samples or 5,33% had the titer of 1/200. By applying classic bacteriological methods, B. canis was isolated from three samples of homogenized reproductive organ tissue (1,33%), 2 from samples originating from males and one specimen originating from a female. One isolate originated from a serologically negative dog. Of the 225 samples assayed using the Bruce-ladder multiplex PCR method, a positive reaction was established in two (0,88%). Protein concentrations were determined prior to the formulation of the indirect ELISA test. The electrophoretic analysis of antigens retrieved using heat and ultrasound, as well as the densitometric quantification, showed that the antigen retrieved using heat had the most prevalent molecular weight fraction of 10,95 kDa with a participation of as much as 43,12% that corresponds to Brucella R LPS. The same fraction was represented in the antigen retrieved using ultrasound with 11,56%, or in a quantity that was 3,7 times lower... Bruceloza pasa je bolest poznata preko četiri decenije, ali i pored toga ne postoje standardizovani dijagnostički protokoli, kao ni generalni dogovor o najprikladnijem dijagnostičkom testu. Svaka laboratorija definiše sopstvene kriterijume. Ovakva raznovrsnost testova i nedostatak jasno definisanih protokola dovodi do teškoća u interpretaciji rezultata seroloških testova u različitim laboratorijama. Iz tog razloga, cilj ove doktorske disertacije obuhvatio je unapređenje dijagnostike primenom preporučenih i novih, sopstveno pripremljenih testova. Jedan od zadataka ove disertacije je bilo i ispitivanje upotrebljivosti Bruce-ladder multiplex PCR metode za ispitivanje kliničkih uzoraka odnosno tkiva uterusa i testisa pasa. Sakupljen je materijal (krv, testisi i materice) od 225 nevlasničkih pasa i to 145 ženki i 80 mužjaka. Dobijeni rezultati su pokazali da je od ukupno 225 ispitanih uzoraka, 33 ili 14,67% krvnih seruma imalo merljiv titar antitela u 2-ME TAT. Najniži ispitivani titar od 1/50 imalo je 13 krvnih seruma ili 5,78%, kod 8 krvnih seruma ustanovljen je titar od 1/100 ili 3,55%, dok je titar od 1/200 imalo 12 uzoraka seruma ili 5,33%. Primenom klasičnih bakterioloških metoda, B. canis je izolovana iz tri uzorka homogenizata tkiva reproduktivnih organa (1,33%) i to iz 2 uzorka poreklom od mužjaka i jednog uzorka poreklom od ženke. Jedan izolat je poticao od serološki negativnog psa. Od 225 uzoraka ispitanih Bruce-ladder multiplex PCR metodom, pozitivna reakcija je ustanovljena kod dva (0,88%). Pre formulacije indirektnog ELISA testa, određivane su koncentracije proteina. Elektroforetska analiza antigena dobijenih toplotom i ultrazvukom kao i denzitometrijska kvantifikacija su pokazale da je u antigenu dobijenom toplotom, najzastupljenija frakcija molekulske mase 10,95 kDa sa učešćem od čak 43,12% koja odgovara R-LPS-u brucela. Ista frakcija je u antigenu dobijenom ultrazvukom bila zastupljena sa 11,56%, odnosno u količini koja je bila 3,7x manja...

Cilj ovog istraživanja je bio ispitivanje sposobnosti formiranja biofilma kod serovarijeteta Salmonella izolovanih iz hrane za životinje, poreklom iz 18 mešaona stočne hrane na području Južnobačkog okruga. Izolati korišćeni u ispitivanju (n=100) izolovani su poreklom iz uzoraka hrane za životinje uzorkovanih tokom dve godine ispitivanja (period 2012-2014). Identifikacija izolata izvršena je na osnovu kulturelnih, biohemijskih i seroloških karakteristika na osnovu preporuka standarda SRPS EN ISO 6579:2008. Potvrda i serološka tipizacija izolata Salmonella izvedena je u Nacionalnoj referentnoj laboratoriji za Salmonella, Shigela, Vibrio cholerae i Yersinia enterocolitica, Instituta za javno zdravlje Srbije „Dr Milan Jovanović Batut“ u Beogradu. Kao kontrolni sojevi, u ispitivanju su korišćene referentne kulture S. Typhimurium ATCC 14028 i S. Enteritidis ATCC 13076. Kao supstrat za formiranje biofilma korišćene su površine od polistirena i nerđajućeg čelika. Sposobnost formiranja biofilma ispitana je primenom sledećih metoda: Congo red agar test, pelikula test, test na mikrotitracionim pločama upotrebom kristal violet boje, i skenirajuća elektronska mikroskopija (SEM). Sva ispitivanja izvedena su upotrebom dve hranljive podloge (tripton soja bujona i Luria Bertani bujona) pri temperaturi inkubacije od 20C i 37C. Iz hrane za životinje izolovani su sledeći serovarijeteti Salmonella: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). Kod četiri izolata nije ustanovljen serovarijetet (Salmonella enterica subspecies enterica (1,3,19:i:-)). Prema rezultatima Congo red agar testa, na temperaturi od 20C, 98% izolata Salmonella ekspresioniralo je jednu ili obe komponente matriksa biofilma i formiralo kolonije RDAR, PDAR i BDAR morfotipova... The aim of this research was the investigation into the biofilm-forming ability of Salmonella serovars isolated from animal feed samples originating from 18 feedproducing facilities located in South Bačka District. The isolates which were tested (n=100) were isolated from feed samples collected in a two-year period (from 2012 to 2014). The identification of the isolates was made based on the cultural, biochemical and serological characteristics, following the recommendations given in the SRPS EN ISO 6579:2008 standard. The conformation and serological typing of Salmonella isolates were done in the National Reference Laboratory for Salmonella, Shigela, Vibrio cholerae and Yersinia enterocolitica, at the Institute of Public Health of Serbia 'Dr Milan Jovanović Batut' in Belgrade. In this study reference cultures of S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 were used as the control strains. Surfaces made of polystyrene and of stainless steel were used as substrates for biofilm formation. The ability of Salmonella to form biofilm was tested with the following methods: Congo red agar test, pellicle test, microtiter plate assay with the use of crystal violet dye, and scanning electron microscopy (SEM). The whole investigation was carried out at two temperatures of incubation (20C and 37C). The following Salmonella serovars were isolated from animal feed: Tennessee (18%), Agona (9%), Montevideo (15%), Enteritidis (12%), Stanleyville (6%), Infantis (12%), Typhimurium (3%), Typhimurium monofazni (1%), Mbandaka (6%), Senftenberg (7%), Jerusalem (2%), Thompson (1%), Amsterdam (1%), Colindale (1%), Dahra (1%), Livingstone (1%). In four isolates serovars were not determined (Salmonella enterica subspecies enterica (1,3,19:i:-)). According to the results of Congo red agar test, when grown at 20C, 98% of the isolates expressed one or both components of the Salmonella biofilm matrix and formed colonies of various morphotypes (RDAR, PDAR and BDAR)...

Cilj rada je bio da se ispita uticaj i stimulativno dejstvo proizvoda na bazi natrijum butirata u ishrani zalučene prasadi na proizvodne parametre i zdravstveno stanje životinja. Ogled je organizovan u kontrolisanim uslovima po ogledno kontrolnim grupama na komercijalnoj farmi svinja Vladimirovac koja posluje u sklopu kompanije Delta Agrar. Ogled na zalučenim prasadima je trajao 54 dana. Prasad korišćena u ogledu podeljena su u 3 grupe (dve ogledne i jedna kontrolna grupa) po 16 prasadi, ukupno 48 životinja. Prasad iz ogleda su bili hibridi rasa veliki jorkšir, danski landras i durok. Prasad su odbijena sa starošću 24±1 dan. Kontrolna grupa prasadi je hranjena smešom bez dodatka oglednog preparata na bazi natrijum butirata. Ogledne grupe (O-I i O-II) su dobijale hranu sa dodatkom natrijum butirata u količini od 3 kg/t odnosno 5 kg/t gotove smeše. Tokom trajanja ogleda praćeni su proizvodni rezultati i zdravstveno stanje prasadi. Ispitivanja su urađena na prasadima oba pola. Na početku ogleda izvršeno je merenje mase zalučene prasadi. Početna masa prasadi je bila 6,56±0,02 kg. U toku trajanja ogleda izvršena su četiri kontrolna merenja i izmerena je utrošena količina hrane. Izvršena je hemijska analiza uzoraka hrane i izračunati su proizvodni rezultati. Na kraju ogleda iz svake grupe je zaklano po 6 prasadi, ukupno 18 životinja, a prilikom klanja uzeti su uzorci creva, sadržaja creva, kao i uzorci jetre i bubrega. U toku trajanja ogleda prasad kontrolne i oglednih grupa bila su klinički zdrava, vitalna i uobičajenog ponašanja. Prosečne telesne mase kao i prirasti prasadi bili su ujednačeni kod kontrolne i oglednih grupa do 33. dana ogleda. Na kraju ogleda prosečne telesne mase, odnosno prosečni prirasti prasadi oglednih grupa bili su statistički značajno veći od prosečnih telesnih masa, odnosno prosečnih prirasta prasadi kontrolne grupe. Ukupna i dnevna konzumacija hrane bila je veća kod kontrolne grupe prasadi u odnosu na ukupnu, odnosno dnevnu konzumaciju oglednih grupa prasadi. Najbolju konverziju imala su prasad O-I grupe, zatim prasad O-II grupe, a najlošiju konverziju su imala prasad kontrolne grupe. Prosečna masa trupa, kao i prosečni randman klanja bili su statistički značajno veći kod oglednih grupa prasadi u odnosu na kontrolnu grupu. Prosečne mase, odnosno prosečne dužine pojedinih segmenata creva, kao i prosečna ukupna masa, odnosno prosečna ukupna dužina creva oglednih grupa bile su statistički značajno veće u odnosu na kontrolnu grupu prasadi, sa izuzetkom prosečne mase kolona i prosečne dužine duodenuma koje su bile statistički značajno veće kod kontrolne grupe prasadi. Između pH vrednosti intestinalnog sadržaja tankog, odnosno debelog creva ispitivanih grupa prasadi nisu utvrđene statistički značajne razlike. U uzorcima sadržaja ileuma, odnosno cekuma kontrolne i oglednih grupa prasadi nisu utvrđene statistički značajne razlike između prosečnih vrednosti ukupnog broja aerobnih mezofilnih bakterija, vrsta Enterococcus i vrsta Lactobacillus... The aim of this study was to investigate the influence of the stimulating effect of products based on sodium butyrate in the diet of weaned piglets on performance and health of the animals. Experiment was organized in controlled conditions (experimental and control groups) on a commercial pig farm Vladimirovac that operates within the company Delta Agrar. Experiment on weaned piglets lasted 54 days. Piglets used in the experiment were divided into 3 groups (two experimental and one control group) of 16 piglets, a total of 48 animals. Piglets in the experiment were hybrids of breeds Large White, Danish Landrace and Duroc. Piglets were weaned at the age of 24±1 day. A control group of piglets was fed with the mixture without the addition of preparation based on sodium butyrate. Experimental groups (O-I and O-II) were fed with the addition of sodium butyrate in an amount of 3 kg/t and 5 kg/t of feed mixture. During the experiment, health and performance of piglets were monitored. Examinations were conducted on piglets of both sexes. At the beginning of the experiment weight of weaned piglets were measured. Starting weight of piglets was 6.56±0.02 kg. During the experiment, it was performed 4 control measurements and a measure of the consumed feed. Chemical analysis of feed samples was conducted and the production results were calculated. At the end of the experiment 6 piglets from each group were slaughtered, a total of 18 animals. After slaughter samples of intestines, intestinal contents, as well as samples of liver and kidney were taken. During the experiment, piglets from control and experimental groups were clinically healthy, vital and with normal behaviour. Average body weight and weight gain of piglets were uniform in the control and experimental groups to 33. day of experiment. At the end of the experiment the average body weight and average weight gain of piglets in experimental groups were significantly higher than the average body weight, and average weight gain of piglets in the control group. The total and daily feed intake were higher in the control group of piglets compared to the total and daily feed intake of the experimental groups of piglets. The best conversion had piglets of O-I group, then piglets of O-II group and the worst feed/gain ratio had piglets of the control group. The average carcass weight and average carcass yield was statistically significantly higher in the experimental groups of piglets compared to the control group. Average weight and average length of individual segments of intestine, as well as the average total weight and average total length of the intestine in the experimental groups of piglets were significantly higher than the control group of piglets, with the exception of the average mass of the colon and the average length of the duodenum, which were significantly higher in the the control group of piglets. Intestine content pH values of the small and large intestine of piglets in examined groups showed no statistically significant differences. In the samples of the content in the ileum and cecum of the piglets in the control group compared to the samples of the content in the ileum and cecum of the piglets in experimental groups were no statistically significant differences between the average values of the total number of aerobic mesophilic bacteria, Enterococcus species and Lactobacillus species...

Класична куга свиња (ККС) је веома контагиозно обољење домаћих и дивљих свиња. Економски посматрано ККС представља највећу претњу свињарској производњи једне земље. У многим земљама, ККС је успешно искорењена применом опсежних мера контроле присуства вируса ККС у природним резервоарима, биосигурносне заштите, нешкодљивог уништавања оболелих свиња, контроле промета животиња, упоредо са програмом масовне вакцинације. Успешно заустављање циркулације вируса ККС кроз пријемчиву популацију и прекидање ланца инфекције уз примену вакцинације, захтева висок ниво имунизације свиња. Ако се вакцинација спроводи систематски на територији целе земље, долази до значајног смањивања клиничких случајева ККС и до заустављања циркулације вируса. Искуства су показала да је ерадикацију уз примену масовне вакцинације могуће постићи у року од годину дана. Да би се обезбедио одговарајући ниво имунитета стада и избегли нежељени ефекти ниског обухвата вакцинацијом, као што је присуство „маскираних“ случајева ККС, потребно је обезбедити константну вакцинацију свиња на терену. Упоредо са имунопрофиласком неопходно је спроводити и мониторинг вакцинације. Мониторинг вакцинације је заснован на плану узорковања, теренском истраживању и препознавању ризичних фактора који условљавају низак обухват имунизацијом. Циљ испитивања је био да се одреди одговарајући поступак израчунавања величине репрезентативног узорка, односно потребног броја насељених места, породичних фарми, сеоских газдинстава и свиња, код којих треба спровести епизоотиолошко истраживање и испитивање сероконверзије, ради одређивања обухвата вакцинацијом против ККС. У раду је описана студија пресека, примерена екстензивним условима држања свиња и урађен прорачун репрезентативног узорка на нивоу популације свиња у Републици Србији. Такође је извршено истраживање обухвата вакцинацијом против ККС на територији три општине у Републици Србији и испитана ефикасност имунизације. Classical swine fever is a highly contagious viral disease of pigs and wild boars. In economic point of view, classical swine fever is one of the highest threats to the pig production. In many countries, classical swine fever has been successfully eradicated with the application of wide control measures such as monitoring of CSFV in natural reservoirs, biosecurity measures, depopulation of diseased pigs and pigs which were in contact with diseased pigs, movement restriction, together with the vaccination of pigs. The efficient control and eradication of CSF by applying vaccination of pigs require a high degree of vaccination coverage in all categories of pigs. Experience shows that CSF can be eradicated in endemic areas with a strict vaccination regime within a year. To ensure satisfactory level of heard immunity and to avoid perverse effects of vaccination like complications due to low coverage, such as the presence of infected undetected pigs, i.e. the presence of "masked" clusters of diseased pigs with a mild clinical picture difficult to differentiate, it is necessary to ensure constant vaccination. Vaccination of pigs should be constantly monitored. Monitoring of vaccination is based on sampling plan of priory estimated required number of samples which have to be tested for the presence of seroconversion, filed investigation and detection of risk factors that may affect the quality and coverage of immunization. The objective of this research was to estimate different epizootiological methods and develop appropriate methodology for statistical calculations of sample size, to compute a representative number of settlements, holdings and pigs that should be investigated in order to determine vaccination coverage in extensive pig farming conditions. In the research was also developed sample size, based on probability distribution, on the level of pig population in the Republic of Serbia and estimated vaccination coverage in three municipalities

Cilј istraživanja bio je da se izvrši izolacija, identifikacija i molekularna karakterizacija, uzročnika tuberkuloze goveda u Vojvodini, kao i ispitivanje osetlјivosti izolovanih sojeva na odabrane antituberkulozne antibiotike koji se koriste za terapiju tuberkuloze lјudi u našoj zemlјi. Pored toga cilj istraživanja bilo je i uporedno ispitivanje osetlјivosti i specifičnosti γ-IFN testa u dijagnostici tuberkuloze sa klasičnim mikrobiološkim metodama izolacije i identifikacije uzročnika. Konačno, na osnovu ispitivanja raširenosti, incidencije i prevalencije oboljenja u zapatima cilj je bila i izrada aktuelne epizootiološke karte raširenosti tuberkuloze goveda u Vojvodini. Za izolaciju bakterija MTB-kompleksa korišćeni su uzorci tkiva (pluća i limfni čvorovi) 49 goveda koja su prilikom izvođenja komparativnog tuberkulinskog ili γ-IFN testa imala pozitivnu reakciju. Kod 40 od ovih grla prilikom patomorfološkog pregleda na liniji klanja, bile su ustanovljene promene koje mogu ukazivati na tuberkulozu. Bakterije MTB-kompleksa uspešno su izolovane iz uzoraka tkiva 19 goveda, dok je nalaz acidorezistentnih bakterija ustanovljen direktnom mikroskopijom u uzorcima limfnih čvorova kod još 3 grla, ali izolacija nije uspela usled kontaminacije. Na osnovu klasičnih bakterioloških metoda, izgleda i rasta kolonija, kao i primenom standardnih biohemijskih testova identifikacije, svi izolati su tipizovani u vrstu M. bovis. Na osetljivost prema izoniazidu, streptomicinu, etambutolu i rifampicinu, antituberkuloticima prve linije koji se u našoj zemlji koriste za lečenje ljudi obolelih od tuberkuloze, standardnom metodom proporcija, ispitano je 5 reprezentativnih izolata. Izolati su bili poreklom od goveda iz pet različitih zapata lociranih u naseljima na teritoriji 5 opština, od kojih su 4 u Južnobačkom i 1 u Sremskom okrugu. Ustanovljena je osetljivost izolata na sve ispitivane antituberkulotike, odnosno ni u jednom slučaju nije utvrđena pojava rezistencije. Metodom PCR spoligotipizacije u cilju molekularne karakterizacije i genotipizacije bakterija M.tuberculosis kompleksa ispitano je 18 izolata mikobakterija kod kojih je proces ekstrakcije DNK bio uspešan. Rezultati molekularne karakterizacije odnosno spoligotipizacije izolovanih mikobakterija pokazuju odsustvo spejsera 1 i niza spejsera od 3 sve do 16, kao i spejsera 28 što je karakteristično za Mycobacterium caprae... The aim of this research was isolation, genotyping and molecular characterization of the causative agent of cattle tuberculosis in the province of Vojvodina, as well as evaluation of susceptibility for isolated zoonotic MTB-complex bacteria towards a panel of selected anti-tuberculosis drugs used in tuberculosis treatment for humans in our country. Additionally, the goal of the research was to estimate sensitivity of γ-IFN test for tuberculosis diagnosis in cattle compared to standard microbiological methods of the isolation or histopathological identification of lesions. Finally, based on the animal incidence and herd prevalence per year, the objective was to create the actual epizootiological map of disease occurrence in Vojvodina province. For the isolation of MTB-complex bacteria tissue samples (lung and lymph nodes) from 49 test positive cattle on single comparative tuberculin test were used. During the post mortem examination at the abattoir, visible lesions were present in 40 animals. MTB-complex bacteria were successfully isolated from the pathological material of 19 bovines, while direct microscopic examination of lymph node smears yielded positive result for acid fast bacteria, in additional three animals, but the isolation failed due to contamination or other reasons. Based on the classic bacteriological methods, growth characteristics, colony morphology and biochemical tests all isolates were designated as M. bovis species. PCR-based spoligotyping, was performed on 18 MTB-complex isolates following successful DNA extraction, and a single spoligotype pattern was identified. The results of the molecular characterization, revealed the absence of spacer 1, spacer region 3-16, as well as spacer 28, in all isolates, a characteristic pattern for Mycobacterium caprae. The absence of spacer 28 and spacer region 39-43 suggests that all isolates belong to single cluster. Automated analysis of the binary code of the submitted spoligotype patterns in two largest online databases came out with identifier number SB 0418 and ST 647 respectively, which represents the predominant Mycobacterium caprae cluster in Central-European countries...

U ovom ispitivanju vršeno je uzorkovanje briseva poreklom od klinički zdravih riba koje su poticale iz različitih sredina (ribnjaci, akvarijumi, riblje pijace). Izvršena je izolacija bakterija koje su sastavni deo mikrobioma kože, škrga i creva riba i ispitivana je osetljivost ovih bakterija na određeni broj antibiotika koji se koriste u veterinarskoj i humanoj medicinskoj praksi. Precizna identifikacija ispitivanih sojeva bakterija vršena je primenom metoda PCR, sekvenciranje gena za 16S rRNK, MALDI-TOF. Primenom disk difuzionog testa i E testa ispitivano je fenotipsko ispoljavanje rezistencije na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, cefalospirine III i IV generacije, aminoglikozide, tetraciklin, kolistin, flurohinolone i hloramfenikol. Prisustvo gena rezistencije, njihova lokalizacija (na hromozomu ili na mobilnim genetičkim elementima) vršena je primenom metode PCR. Kod sojeva koji su ispoljili rezistenciju na nabrojane antibiotike traženi su plazmidi i ispitivana je konjugabilnost izolovanih plazmida. Posmatrano na ukupan broj ispitanih sojeva u ovom istraživanju, bez obzira na rod i vrstu bakterija, ukupno je nađeno 55% sojeva koji su bili osetljivi na sve antibiotike, kod 22,8% sojeva nađena je rezistencija na 3 do 16 antibiotika uključujući i antibiotike koji se koriste isključivo kod ljudi (karbapenemi, ureidopenicilini, cefalosporini III i IV generacije). Ukupno 22,2% sojeva bilo je rezistentno na 1 do 2 antibiotika, mada je i među tim sojevima bilo onih koji su bili rezistentni na antibiotike registrovane samo za upotrebu kod ljudi (ceftazidim, piperacilin). Kod soja A. hydrophila izolovanom iz akvarijumske ribice gupi potvrđen je mehanizam rezistencije nalazom gena rmtB koji je bio lokalizovan na transpozonu Tn1548 smeštenom na konjugabilnom plazmidu koji je po tipu replikona bio kategorisan u grupu IncL/M. Kod sojeva Pseudomonas koji su bili rezistentni na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, kao i na cefalosporine III i IV generacije, nisu nađeni geni za, karbapenemaze, MßL, ESBL, OXA i AmpC beta-laktamaze (KPC, OXA-23, OXA- 24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTXM- 9, OXA-1, OXA-9, AmpC grupni kao i pojedinačni-MOXM, CITM, ACCM, EBCM, FOXM, DHAM).Na osnovu dobijenih rezultata primenom E testa, kod 3 soja iz roda Pseudomonas izolovanih od šarana nađena je rezistencija na kolistin sa dobijenim vrednostima MIK 4 μg/mL. During this research, a series of microbiological smears was collected from clinically healthy fish found in different environments (aquaculture ponds, aquariums, and fish markets) has been done. Bacteria which belong to skin microbiome, gills, and fish intestines have been isolated, and their sensitivity to several antibiotics used in veterinary and human practice has been tested. Precise identification of tested strains of bacteria has been performed with PCR method, 16S rRNA gene sequencing and MALDI-TOF. Phenotypical manifestation of resistance to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosporins of the third and fourth generation, aminoglycosides, tetracyclines, colistin, fluoroquinolones and chloramphenicol has been tested using disc diffusion method and E test. Presence of resistant genes, their localization (on chromosome or on mobile genetic elements) has been conducted with PCR method. For strains showing resistance to the antibiotics mentioned above, plasmids have been searched and conjugation of isolated plasmids has been tested. Observing the total number of tested strains in this research, regardless of the genus and species of bacteria, 55% of examined strains were found to be sensitive to all antibiotics, and in 22.8% of strains resistance was observed to 3-16 antibiotics, including antibiotics used exclusively in human medicine (carbapenems, ureidopenicillins, cephalosporins of third and fourth generation). In additional 22.2% of strains the resistance to 1 or 2 antibiotics was recorded, including resistance to antibiotics registered for exclusive use in human medicine (ceftazidime, piperacillin). In A. hydrophila strain isolated from aquarium fish guppy that showed resistance to all 16 antibiotics, a mechanism of resistance has been confirmed by identifying gene rmtB which has been localized on transposon Tn1548 located on conjugal plasmid which belongs to group IncL/M type of replicon. In Pseudomonas strains resistant to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosprins of third and fourth generation, the genes for carbapenemases MßL, ESBL, OXA and AmpC beta-laktamases (KPC, OXA-23, OXA-24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTX-M-9, OXA-1, OXA-9, group AmpC and specific MOXM, CITM, ACCM, EBCM, FOXM, DHAM) have not been found. Based on results obtained with use of E test, resistence to colistin has been found in 3 strains of Pseudomonas spp. isolated from carp with MIC values of 4 μg/mL.

Osnovni cilj istraživanja ove doktorske disertacije je bio da se ispita uticaj ishrane brojlera obrocima sa dodatim različitim fitogenim aditivima na zdravstveno stanje, proizvodne rezultate (tjelesna masa, prirast, konzumacija i konverzija hrane), parametre prinosa (randman i procenat mesa u trupu) i kvaliteta mesa brojlera (hemijski sastav i senzorne anlize), mikrobiološke i morfološke karakteristike pojedinih segmenata crijeva, kao i opravdanost korištenja fitogenih aditiva u ishrani brojlera. Ogled je izveden na ukupno 240 brojlera provenijencije Cobb 500, podijeljenih u 4 grupe po 60 jedinki. Hranjeni su standardnim smješama po preporuci proizvođača, s tim što su se grupe jedino razlikovale u tome što su ogledne grupe u smješi za ishranu imale dodate različite komercijalne fitogene aditive (O-I grupa dvokomponentni fitogeni aditiv- timol i cinamaldehid u količini od 100 g/t, O-II grupa četverokomponentni fitogeni aditiv-esencijalna ulja kima, nane, karanfilića i anisa u količini od 150 g/t i O-III grupa jednokomponentni fitogeni aditiv-timol u količini od 750 g/t) i kontrolna grupa bez dodataka fitogenog aditiva. Smješe za ishranu su bile izbalansirane i u potpunosti zadovoljavale potrebe brojlera u svim fazama tova. Za vrijeme trajanja ogleda su praćeni proizvodni parametri (tjelesna masa, prirast, konzumacija i konverzija hrane) i zdravstveno stanje. Kontrolna mjerenja tjelesne mase i konzumacije hrane vršena su na kraju svake faze tova. Na kraju tova brojleri su izmjereni, izračunata je potrošena količina hrane, a na klaonici je izvršena primarna obrada, hlađenje, mjerenje mase ohlađenih trupova, rasijecanje, mjerenje mase osnovnih dijelova trupa (bataka sa krabatakom i grudi) i uzimanje uzoraka mesa za hemijske analize (hemijski sastav mesa) i senzorne analize i uzoraka crijeva za mikrobiološke i histološke analize. Korištenjem fitogenih aditiva su postignuti bolji proizvodni rezultati oglednih grupa u odnosu na kontrolnu grupu. Tjelesne mase brojlera oglednih grupa su bile veće od kontrolne grupe u svim fazama tova. U periodu tova do 10. dana, utvrđeno je da je prosječna tjelesna masa brojlera kontrolne grupe statistički značajno manja od brojlera O-I i O-III grupe. U periodu tova do 20. i 42. dana, utvrđene su statitički značajne razlike između posmatranih oglednih grupa u odnosu na kontrolnu grupu. U svim fazama tova, kao i za cijeli period tova ogledne grupe su imale veći prosječni prirast od kontrolne grupe. Na kraju tova najveći prirast je imala O-III grupa, a statistički značajne razlike su utvrđene između posmatranih oglednih grupa u odnosu na kontrolnu grupu. Ukupna konzumacija hrane za cijeli period tova je bila najveća kod kontrolne grupe, a najmanja kod O-I grupe... The aim of this doctoral thesis was to investigate the influence of broiler diet with addition of different phytogenic additives on the health status, production results (body weight, weight gain, consumption and feed conversion), parameters yields (yields and percentage of meat in the carcass), quality of broiler meat (chemical composition and sensory analysis), microbiological and morphological characteristics of individual segments of the intestine, as well as the explanation for the use of phytogenic additives in broilers diet. The experiment was conducted on a total of 240 broilers Cobb 500, divided into four groups of 60 individuals. They were fed by standard mixtures recommended by the manufacturer, taking into account that the groups differed only in the fact that the experimental group was fed with mixture with different commercial phytogenic additives (O-I group of two-component phytogenic additive- thymol and cinnamaldehyde in the amount of 100 g / t, O-II group four-component phytogenic additive-essential oils of caraway, mint, clove and anise in the amount of 150 g / t and O-II group of one phytogenic additive-thymol in the amount of 750 g / t) and the control group without phytogenic additives. Mixtures were balanced to completely satisfied the diet needs of broilers at all stages of fattening. Production parameters were monitored during the experiment (body weight, weight gain, consumption and feed conversion) as well as a health condition. Control measurements of body weight and feed consumption were carried out at the end of each stage of fattening. At the end of the fattening broilers were measured, the amount of feed consumed was calculated, and primary processing was carried out in the slaughter- cooling, measuring the mass of chilled carcasses, cutting, measuring the mass of major carcass parts (thighs with drumsticks and breasts) and taking meat samples for chemical analysis (chemical composition of meat) and sensory analysis and intestines' samples for microbiological and histological analysis. Better production results of experimental groups compared to the control group have been achieved by using the phytogenic additive. Body weight of broilers of experimental groups was higher than the control group at all stages of fattening. During the ten days fattening period, the average body weight of the broiler from the control group was significantly lower than broilers from O-I and O-III group. During the 20 and 42 days fattening period, significant differences were determined between the experimental groups compared to the control group...

Salmonele su najčešći prouzrokovači.crevnihinfekcija kod ljudi i životinja Široko surasprostranjene u prirodi a mogu kolonizovatirazličite domaćine Poebno su značajne oneživotinje čije se meso koristi za ishranu ljudi, akoje su i glavni izvor infekcije ljudi. Inficiraneživotinje ne ispoljavaju simptome i terapija sesprovodi nalazom salmonela prilikom rutinskekontrole zdravstvenog stanja. Posebno, kod ljudi iživotinja salmoneloze su značajne i zbogkliconoštva koje može trajati veoma dugo zavisnood starosti inficiranog organizma. Čest su uzrokalimentarnih toksiinfekcija kod ljudi. Duž čitavoglanca ishrane moguća je i sekundarnakontaminacija salmonelama. Pored rutinskihmikrobioloških analiza u otkrivanju izvora iputeva širenja infekcije koriste se i molekularnemetode koje daju precizne podatke o klonalnomporeklu bakterija izolovanih iz obolelih ljudi,namirnica i životinja. Međunarodnom trgovinomhrane isti tipovi bakterija mogu se pojaviti nageografski udaljenim lokacijama. Molekularnakarakterizacija Salmonella je značajna zbogodređivanja raznolikosti sojeva. Potrebno je da seizolati tipiziraju ne samo do nivoa vrste i serotipanego i preciznije. Tipizacija je bitna za utvrđivanjeepidemiološke povezanosti izolata, agenotipizacija podrazumeva direktnu analizuDNK. Geni rezistencije koji se od saprofita mogupreneti na patogene vrste imaju važnu ulogu upojavljivanju rezistentnih i multirezistentnihsojeva. Međusobni odnos gena rezistencije iupotrebe antimikrobnih sredstava određujejednačinu rezistencije na antimikrobna sredstva.Rezistencija predstavlja problem iako jesalmoneloza samolimitirajuća infekcija zbog čegase terapija antibioticima primenjuje samo koddece, starijih ljudi i u slučajevima sistemskihinfekcija. Navedena problematika je aktuelna i uCrnoj Gori jer ne postoji stalan monitoringprimene antimikrobnih sredstava kod domaćihživotinja. Iz navedenih razloga cilj ove doktorskedisertacije bilo je utvrđivanje prisustva Salmonellavrsta na farmama živine sa tri lokaliteta u CrnojGori, izolacija i identifikacija primenom standardnihmikrobioloških metoda, serotipizacija pomoćuaglutinacije na predmetnom staklu (slideagglutination) uz primenu anti-O i anti-H seruma.(Staten Serum Institute, Danska). Primenomstandardnih mikrobioloških metoda utvrđeno jeprisustvo serovarijeteta Salmonella Enteritidis iSalmonella Tiphymurium. SerovarijetetiSalmonella .Gallinarum biotip Gallinarum iSalmonella Gallinarum biotip Pullorum nisu bileserološki tipizirane. Identifikacija navedenihserovarijeteta je potvrđena metodom multipleksPCR detekcijom amplifikovanih DNK fragmenatau agaroznom gelu.. Nakon digestije 50 izolataSalmonella enterica podvrste enterica odabranihprema lokalitetima i zastupljenosti pomoću SpeIrestrikcionog enzma i njihovom analizomprimenom PFGE utvrđeno je 5 različitih SpeIpulsotipova. Kod 10% ispitivanih sojevaustanovljena je rezistencija na tetraciklin istreptomicin. Svi ispitivani serovarijetetisalmonela bili su osetljivi na amoksicilin saklavulanskom kiselinom, enrofloksacin,ciprofloksacin, sulfametoksazol-trimetoprim,cefuroksim, ceftriakson i norfloksacin. VrednostiMIK (minimalnih inhibitornih koncentracija)određenih antibiotika za odabrane sojeveserovarijeteta Salmonella Enteritidis primenomaparata VITEK 2 iznosile su za: piperacilin ≤ 4 –64 μg/ml (S – R) , cefuroksim 4 – 32 μg/ml (S -R), cefuroksim aksetil 4 –32 μg/ml (S– R),cefiksim ≤ 0,25 – 2 μg/ml (S - I), ceftriakson ≤ 1 –2 μg/ml (S - I) i minociklin ≤ 1 – 4 μg/ml (S),tetraciklin ≤ 1 μg/ml (S), tigeciklin ≤ 0,5 – 1 μg/ml(S), hloramfenikol ≤ 2 – 8 μg/ml (S), kolistin ≤0,5–1 μg/ml (S) i sulfametoksazol/trimetoprim ≤0,5 μg/ml (S)., Salmonella is the most common cause of alimentarytoxic infections among humans. They have beenadapted to a number of warm-blooded animals. Theinfected animals do not exhibit symptoms and thetreatment performs by finding of salmonella in routinehealth check. The secondary contamination bysalmonella ois possible in during entire food chain.Apart from the routine microbiological analysis indetection of sources and pathways of spreading theinfection, there are also used the molecular methodsthat provide accurate information about the clonalorigin of bacteria isolated from diseased humans, foodand animals. During international trade of food, thesame types of bacteria can occur in geographicallyremote locations. Molecular characterization ofSalmonella is important in determination of diversity ofstrains. It is necessary isolates to be typified not only tothe level of species and serotypes but also moreprecisely. Typification is essential to determine theepidemiological connection of isolates. Genotypingincludes a direct analysis of DNA. The resistance genesthat can be transferred from saprophytes to pathogenicmicroorganisms play an important role in theemergence of resistant and multiresistant strains. Theabove mentoned is also a current issue in our countrybecause there is a constant monitoring of using ofantimicrobials drugs to farm animals. For these reasons,the aim of this dissertation is to examine the serovars ofSalmonella in Montenegro, their molecularcharacterization using biomolecular methods based onisolation of DNA and subsequent amplification ofserovar-specific genes.(multiplex PCR method andPFGE), and testing sensitivity, or resistance toantimicrobial drugs used in clinic vet practice.

Salmonele su najčešći prouzrokovači.crevnih infekcija kod ljudi i životinja Široko su rasprostranjene u prirodi a mogu kolonizovati različite domaćine Poebno su značajne one životinje čije se meso koristi za ishranu ljudi, a koje su i glavni izvor infekcije ljudi. Inficirane životinje ne ispoljavaju simptome i terapija se sprovodi nalazom salmonela prilikom rutinske kontrole zdravstvenog stanja. Posebno, kod ljudi i životinja salmoneloze su značajne i zbog kliconoštva koje može trajati veoma dugo zavisno od starosti inficiranog organizma. Čest su uzrok alimentarnih toksiinfekcija kod ljudi. Duž čitavog lanca ishrane moguća je i sekundarna kontaminacija salmonelama. Pored rutinskih mikrobioloških analiza u otkrivanju izvora i puteva širenja infekcije koriste se i molekularne metode koje daju precizne podatke o klonalnom poreklu bakterija izolovanih iz obolelih ljudi, namirnica i životinja. Međunarodnom trgovinom hrane isti tipovi bakterija mogu se pojaviti na geografski udaljenim lokacijama. Molekularna karakterizacija Salmonella je značajna zbog određivanja raznolikosti sojeva. Potrebno je da se izolati tipiziraju ne samo do nivoa vrste i serotipa nego i preciznije. Tipizacija je bitna za utvrđivanje epidemiološke povezanosti izolata, a genotipizacija podrazumeva direktnu analizu DNK. Geni rezistencije koji se od saprofita mogu preneti na patogene vrste imaju važnu ulogu u pojavljivanju rezistentnih i multirezistentnih sojeva. Međusobni odnos gena rezistencije i upotrebe antimikrobnih sredstava određuje jednačinu rezistencije na antimikrobna sredstva. Rezistencija predstavlja problem iako je salmoneloza samolimitirajuća infekcija zbog čega se terapija antibioticima primenjuje samo kod dece, starijih ljudi i u slučajevima sistemskih infekcija. Navedena problematika je aktuelna i u Crnoj Gori jer ne postoji stalan monitoring primene antimikrobnih sredstava kod domaćih životinja. Iz navedenih razloga cilj ove doktorske disertacije bilo je utvrđivanje prisustva Salmonella vrsta na farmama živine sa tri lokaliteta u Crnoj Gori, izolacija i identifikacija primenom standardnih mikrobioloških metoda, serotipizacija pomoću aglutinacije na predmetnom staklu (slide agglutination) uz primenu anti-O i anti-H seruma .(Staten Serum Institute, Danska). Primenom standardnih mikrobioloških metoda utvrđeno je prisustvo serovarijeteta Salmonella Enteritidis i Salmonella Tiphymurium. Serovarijeteti Salmonella .Gallinarum biotip Gallinarum i Salmonella Gallinarum biotip Pullorum nisu bile serološki tipizirane. Identifikacija navedenih serovarijeteta je potvrđena metodom multipleks PCR detekcijom amplifikovanih DNK fragmenata u agaroznom gelu.. Nakon digestije 50 izolata Salmonella enterica podvrste enterica odabranih prema lokalitetima i zastupljenosti pomoću SpeI restrikcionog enzma i njihovom analizom primenom PFGE utvrđeno je 5 različitih SpeI pulsotipova. Kod 10% ispitivanih sojeva ustanovljena je rezistencija na tetraciklin i streptomicin. Svi ispitivani serovarijeteti salmonela bili su osetljivi na amoksicilin sa klavulanskom kiselinom, enrofloksacin, ciprofloksacin, sulfametoksazol-trimetoprim, cefuroksim, ceftriakson i norfloksacin. Vrednosti MIK (minimalnih inhibitornih koncentracija) određenih antibiotika za odabrane sojeve serovarijeteta Salmonella Enteritidis primenom aparata VITEK 2 iznosile su za: piperacilin ≤ 4 – 64 μg/ml (S – R) , cefuroksim 4 – 32 μg/ml (S - R), cefuroksim aksetil 4 –32 μg/ml (S– R), cefiksim ≤ 0,25 – 2 μg/ml (S - I), ceftriakson ≤ 1 – 2 μg/ml (S - I) i minociklin ≤ 1 – 4 μg/ml (S), tetraciklin ≤ 1 μg/ml (S), tigeciklin ≤ 0,5 – 1 μg/ml (S), hloramfenikol ≤ 2 – 8 μg/ml (S), kolistin ≤ 0,5–1 μg/ml (S) i sulfametoksazol/trimetoprim ≤ 0,5 μg/ml (S). Salmonella is the most common cause of alimentary toxic infections among humans. They have been adapted to a number of warm-blooded animals. The infected animals do not exhibit symptoms and the treatment performs by finding of salmonella in routine health check. The secondary contamination by salmonella ois possible in during entire food chain. Apart from the routine microbiological analysis in detection of sources and pathways of spreading the infection, there are also used the molecular methods that provide accurate information about the clonal origin of bacteria isolated from diseased humans, food and animals. During international trade of food, the same types of bacteria can occur in geographically remote locations. Molecular characterization of Salmonella is important in determination of diversity of strains. It is necessary isolates to be typified not only to the level of species and serotypes but also more precisely. Typification is essential to determine the epidemiological connection of isolates. Genotyping includes a direct analysis of DNA. The resistance genes that can be transferred from saprophytes to pathogenic microorganisms play an important role in the emergence of resistant and multiresistant strains. The above mentoned is also a current issue in our country because there is a constant monitoring of using of antimicrobials drugs to farm animals. For these reasons, the aim of this dissertation is to examine the serovars of Salmonella in Montenegro, their molecular characterization using biomolecular methods based on isolation of DNA and subsequent amplification of serovar-specific genes.(multiplex PCR method and PFGE), and testing sensitivity, or resistance to antimicrobial drugs used in clinic vet practice.

Virus Zapadnog Nila (West Nile virus-WNV) je izolovan 1937. godine u West Nile distrihtu u Ugandi, po čemu je i dobio ime. Pripada familiji Flaviviridae i rodu Flavivirus. Prenosi se putem insekata-komaraca pa se svrstava u grupu Arbovirusa, a ptice predstavljaju prave domaćine i rezervoare virusa u prirodi. Čovek i sisari su slučajni domaćini koji obolevaju sa kliničkom slikom neuroinvazivnog oboljenja. Brojne epidemije koje je izazvao su bile praćene velikim brojem obolelih ljudi i životinja i materijalnim štetama. Serološka laboratorijska dijagnostika infekcije WNV je složena zbog unakrsnih reakcija sa drugim virusima iz iste familije (Japanski encefalitis, krpeljski encefalitis, Usutu i dr.), ali i odabira dijagnostičkog metoda koji će dati najoptimalnije rezultate. Cilj istraživanja je bio da se pripreme „in house“ testovi (AGID, IFA i ELISA) za serološku dijagnostiku WNV i da se uporede dobijeni rezultati „in house“ testova sa rezultatima dobijenih primenom komercijalno dostupnih testova u cilјu određivanja osetlјivosti i specifičnosti upotreblјenih testova radi utvrđivanja koji od primenjenih testova su najpogodniji za otkrivanje infekcije WNV. Primenom pomenutih testova utvrđena je seroprevalencija u populacijama ispitivanih vrsta (konji, lјudi, psi) na odabranim lokalitetima Republike Srbije, što omogućava mapiranje ugroženih područja i definisanje mera kontrole i suzbijanja infekcije. Ispitano je ukupno 153 krvna seruma ljudi, 303 krvna seruma konja i 184 krvna seruma pasa. Uzorci su prikupljeni u periodu od 2011.-2013. godine na ukupno 15 različitih lokaliteta- regiona Republike Srbije. Ustanovljena seroprevalencija je varirala u odnosu na lokalitet sa većom vrednošću u regionima u slivu Save i Dunava. Pripremljena su tri „in house“ testa (AGID, IFA i ELISA), a rezultati testova su upoređeni sa rezultatima komercijalno dostupnih testova. Statističkim poređenjem primenom McNemarove metode nisu utvrđene statistički značajne razlike između „in house“ IFA testa i komercijalno dostupnog testa na uzorcima poreklom od ljudi i konja... West Nile virus (WNV) was discovered in West Nile district of Uganda in 1937., and named after place of discovery. It is classified in fam. Flaviviridae and genus Flavivirus. WNV is transmitted via insects-mosquitos and it is a member of Arbovirus group. Birds are true hosts and reservoirs of virus in nature. Humans and other mammals are accidental hosts showing clinical signs of neuroinvasive disease. Numerous epidemics caused by WNV were followed by large number of affected people and animals and significant economical loses. Serological laboratory diagnostics is difficult due to cross reactions with other viruses from the same family (Japanese encephalitis virus, tick born encephalitis virus, Usutu etc), and right choice of diagnostic method which would give the most satisfactory results. The goal of research was to prepare „in house“ tests (AGID, IFA and ELISA) for serological diagnostics of WNV, to compare the obtained results of „in house“ and commercially available test in order to determinate sensitivity and specificity of „in house“ tests and the most satisfactory tests in diagnostic of WNV infection. The seroprevalence was determined by use of prepared and available test in populations of horse, dog and human in investigated chosen regions of the Republic of Serbia, which enabled the mapping of endangered areas and definition of control and eradication measures. Total of 153 human blood sera, 303 horse sera and 184 dog sera were collected in period from 2011. to 2013. in 15 different localities of the Republic of Serbia. Determined seroprevalence varies in localities with higher value in localities near Sava and Danube river. Three „in house“ test were made (AGID, IFA and ELISA) and the results were compared to results of commercially available test by use of McNemar test. The statistically significant differences were not established between „in house“ and commercial IFA test in horse and human samples. The use of IFA tests was not possible in dogs due to unspecific reaction...

During this research, a series of microbiological smears was collected from clinically healthy fish found in different environments (aquaculture ponds, aquariums, and fish markets) has been done. Bacteria which belong to skin microbiome, gills, and fish intestines have been isolated, and their sensitivity to several antibiotics used in veterinary and human practice has been tested. Precise identification of tested strains of bacteria has been performed with PCR method, 16S rRNA gene sequencing and MALDI-TOF. Phenotypical manifestation of resistance to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosporins of the third and fourth generation, aminoglycosides, tetracyclines, colistin, fluoroquinolones and chloramphenicol has been tested using disc diffusion method and E test. Presence of resistant genes, their localization (on chromosome or on mobile genetic elements) has been conducted with PCR method. For strains showing resistance to the antibiotics mentioned above, plasmids have been searched and conjugation of isolated plasmids has been tested. Observing the total number of tested strains in this research, regardless of the genus and species of bacteria, 55% of examined strains were found to be sensitive to all antibiotics, and in 22.8% of strains resistance was observed to 3-16 antibiotics, including antibiotics used exclusively in human medicine (carbapenems, ureidopenicillins, cephalosporins of third and fourth generation). In additional 22.2% of strains the resistance to 1 or 2 antibiotics was recorded, including resistance to antibiotics registered for exclusive use in human medicine (ceftazidime, piperacillin). In A. hydrophila strain isolated from aquarium fish guppy that showed resistance to all 16 antibiotics, a mechanism of resistance has been confirmed by identifying gene rmtB which has been localized on transposon Tn1548 located on conjugal plasmid which belongs to group IncL/M type of replicon. In Pseudomonas strains resistant to carbapenems, ureidopenicillins (with and without inhibitors of beta-lactamase), cephalosprins of third and fourth generation, the genes for carbapenemases MßL, ESBL, OXA and AmpC beta-laktamases (KPC, OXA-23, OXA-24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTX-M-9, OXA-1, OXA-9, group AmpC and specific MOXM, CITM, ACCM, EBCM, FOXM, DHAM) have not been found. Based on results obtained with use of E test, resistence to colistin has been found in 3 strains of Pseudomonas spp. isolated from carp with MIC values of 4 μg/mL. U ovom ispitivanju vršeno je uzorkovanje briseva poreklom od klinički zdravih riba koje su poticale iz različitih sredina (ribnjaci, akvarijumi, riblje pijace). Izvršena je izolacija bakterija koje su sastavni deo mikrobioma kože, škrga i creva riba i ispitivana je osetljivost ovih bakterija na određeni broj antibiotika koji se koriste u veterinarskoj i humanoj medicinskoj praksi. Precizna identifikacija ispitivanih sojeva bakterija vršena je primenom metoda PCR, sekvenciranje gena za 16S rRNK, MALDI-TOF. Primenom disk difuzionog testa i E testa ispitivano je fenotipsko ispoljavanje rezistencije na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, cefalospirine III i IV generacije, aminoglikozide, tetraciklin, kolistin, flurohinolone i hloramfenikol. Prisustvo gena rezistencije, njihova lokalizacija (na hromozomu ili na mobilnim genetičkim elementima) vršena je primenom metode PCR. Kod sojeva koji su ispoljili rezistenciju na nabrojane antibiotike traženi su plazmidi i ispitivana je konjugabilnost izolovanih plazmida. Posmatrano na ukupan broj ispitanih sojeva u ovom istraživanju, bez obzira na rod i vrstu bakterija, ukupno je nađeno 55% sojeva koji su bili osetljivi na sve antibiotike, kod 22,8% sojeva nađena je rezistencija na 3 do 16 antibiotika uključujući i antibiotike koji se koriste isključivo kod ljudi (karbapenemi, ureidopenicilini, cefalosporini III i IV generacije). Ukupno 22,2% sojeva bilo je rezistentno na 1 do 2 antibiotika, mada je i među tim sojevima bilo onih koji su bili rezistentni na antibiotike registrovane samo za upotrebu kod ljudi (ceftazidim, piperacilin). Kod soja A. hydrophila izolovanom iz akvarijumske ribice gupi potvrđen je mehanizam rezistencije nalazom gena rmtB koji je bio lokalizovan na transpozonu Tn1548 smeštenom na konjugabilnom plazmidu koji je po tipu replikona bio kategorisan u grupu IncL/M. Kod sojeva Pseudomonas koji su bili rezistentni na karbapeneme, ureidopeniciline sa i bez inhibitora betalaktamaza, kao i na cefalosporine III i IV generacije, nisu nađeni geni za, karbapenemaze, MßL, ESBL, OXA i AmpC beta-laktamaze (KPC, OXA-23, OXA- 24, OXA-40, OXA-58, VIM, IMP, SPM, GIM, NDM, TEM, SHV, CTX-M-1, CTXM- 9, OXA-1, OXA-9, AmpC grupni kao i pojedinačni-MOXM, CITM, ACCM, EBCM, FOXM, DHAM).Na osnovu dobijenih rezultata primenom E testa, kod 3 soja iz roda Pseudomonas izolovanih od šarana nađena je rezistencija na kolistin sa dobijenim vrednostima MIK 4 μg/mL.

U ovom ispitivanju korišćeno je 49 izolata stafilokoka poreklom od hospitalizovanih pacijenata i bolničkog okruženja, od bolesnih životinja, iz uzoraka mleka krava sa klinički manifestnim mastitisom, iz sireva i uzoraka hrane za životinje. Za izolaciju stafilokoka korišćene su standardne mikrobiološke metode, a za identifikaciju stafilokoka do vrste korišćene su komercijalne metode ID32 STAPH (bioMérieux, Francuska) i BBL Crystal Gram-Positive ID Kit (Becton Dickinson, SAD), a za određeni broj izolata korišćen je Vitek MS (bioMérieux, Marcy l’Etoile, Francuska).Takođe u ovom ispitivanju korišćena je i hromogena podloga (chromIDtmMRSA, bioMérieux, Francuska) za brzu detekciju sojeva MRSA od hospitalizovanih ljudi i bolničkog okruženja. Ispitivanje osetljivosti izolovanih vrsta stafilokoka na odabrane antibiotike vršeno je disk difuzionom metodom, a dobijeni rezultati su interpretirani prema preporukama CLSI (Clinical and Laboratory Standards Institute) iz 2008. godine za oksacilin i vankomicin i prema preporukama CLSI iz 2014. godine za cefoksitin i druge antibiotike. Za kontrolu kvaliteta izvođenja ove metode korišćen je referentni soj S. aureus ATCC 25923. Ispitivanje osetljivosti stafilokoka na oksacilin i cefoksitin vršeno je i mikrodilucionom metodom u bujonu prema preporukama NCCLS (National Committee for Clinical Laboratory Standards - sada CLSI) iz 2003. godine, a rezultati su interpretirani prema preporukama CLSI iz 2014. godine, s tim što su rezultati ispitivanja osetljivosti koagulaza-negativnih stafilokoka na cefoksitin interpretirani prema preporukama za S. aureus i S. lugdunensis. Za kontrolu kvaliteta izvođenja ove metode korišćen je referenti soj S. aureus ATCC 29213. Za ispitivanje prisustva penicilinvezujućeg proteina 2a (PBP2a ili PBP2’), korišćen je Slidex®MRSA Detection test (bioMérieux, Francuska), a za detekciju mecA gena kod izolovanih vrsta stafilokoka korišćena je metoda PCR (Polymerase Chain Reaction). Za kontrolu kvaliteta izvođenja ovih reakcija su korišćeni referentni sojevi i to meticilin-rezistentan S. aureus ATCC 43300 kao pozitivna kontrola, a kao negativna kontrola S. aureus ATCC 25923... The investigation comprised 49 isolates of staphylococci originating from hospitalized patients and hospital environment, diseased animals, dairy samples from cows with clinically manifested mastitis, cheeses and feed samples. In the isolation of staphylococci the standard microbiological methods were used; whereas for the identification of staphylococci up to the level of species the following commercial methods were used:ID 32 STAPH (bioMerieux, France) and BBL Crystal Gram-Positive ID Kit (Becton Dickinson, USA), and for certain isolates Vitek MS (bioMérieux, Marcy l'Etoile, France) was used. In addition, the chromogenic medium (chromIDtmMRSA, bioMérieux, France) was used for the rapid detection of MRSA strains from the hospitalized patients and hospital environment. In order to test the susceptibility of the isolated staphylococci to the chosen antibiotics, the disk diffusion test was used, and the obtained results interpreted in accordance with CLSI (Clinical and Laboratory Standards Institute) recommendations from 2008 for oxacillin and vancomycin, and those for cefoxitin and the remaining antibiotics in accordance with the CLSI recommendations from 2014. The reference strain S. aureus ATCC 25923 was used for the testing of the quality of the method used. The susceptibility testing of staphylococci to oxacillin and cefoxitin was done also using the broth microdilution method in accordance with the NCCLS (National Committee for Clinical Laboratory Standards - now CLSI) recommendations from 2003, and the obtained results interpreted using the CLSI recommendations from 2014; the results obtained for the susceptibility of coagulasenegative staphylococci (CoNS) to cefoxitin were interpreted using the recommendations for S. aureus and S. lugdunensis...

Sаvrеmеni nаĉin ţivljenja dоvео је dо pоvеćаnе pоtrаţnjе zа prоizvоdimа оd mеsа kојi оsim tоplоtnе оbrаdе nе zаhtеvајu dоdаtnu priprеmu u dоmаćinstvu. Jedan od od takvih proizvoda je i marinirano meso. Mariniranje pruţa mnoge prednosti, kako za proizvoĊaĉe, tako i za potrošaĉe. Tradicionalno se meso marinira u cilju poboljšanja ukusa, konzistencije i odrţivosti mesa. Kakvi će biti efekti mariniranja zavisi od vrste marinade, ali i kvaliteta mesa kao sirovine. Industrijsko mariniranje ima za cilj, pre svega, poboljšanje tehnoloških i senzornih osobina mesa. U tu svrhu se najĉešće koriste alkalne marinade pripremljene od vode, kuhinjske soli i fosfata. Ove marinade povećavajući pH vrednost mesa omogućavaju veće vezivanje vode u mišićnom tkivu, što konaĉno dovodi do povećanja prinosa i soĉnosti mesa. Sa druge strane, fosfatne soli koje se koriste za pripremu industrijskih marinada ne smatraju se antimikrobnim sredstvima. Zato se sve veća paţnja posvećuje ispitivanjima uticaja razliĉitih fosfata i njihovih kombinacija sa solima organskih kiselina i drugim sastojcima na mikrobiološki status i kvalitet mesa. Pileće meso, u odnosu na druge vrste mesa, ima mnoge prednosti. Ipak, pileće meso je i jedan od najĉešće registrovanih izvora izbijanja salmoneloza kod ljudi, a kao uzroĉnici trovanja najĉešće se navode serotipovi Salmonella Enteritidis i Salmonella Typhimurium. Osim toga, jedini propisima definisan kriterijum bezbednosti za poluproizvode od mesa, kakvo je marinirano pileće meso, je odsustvo Salmonella spp. Zato je nauĉno i struĉno opravdano što je cilj ovog istraţivanja bio da se ispita uticaj mariniranja na rast i preţivljavanje Salmonella vrsta u mesu brojlera. TakoĊe, ispitivanja u okviru ove disertacije imala su za cilj da pokaţu uticaj mariniranja na mikrobiološke pokazatelje higijenskog statusa i odrţivosti mesa, kao i na hemijske i fiziĉko- hemijske parametre kvaliteta mesa brojlera... Contemporary lifestyle has led to increased demand for meat products, that beside heat treatment does not require additional preparation in the household. One such product is a marinated meat. Marinating provides many benefits for both producers and consumers. Traditionally, meat has been marinated in order to improve the taste, consistency and shelf- life of the meat. What will be the effects of marination depends on the type of marinade and the quality of meat as a raw material as well. Industrial marination aims, above all, to improve the technological and sensory characteristics of meat. For this purpose, the most commonly used are alkaline marinades prepared from water, table salt and phosphate. Due to an increase in the pH value of meat, these marinades allow greater water uptake in muscle, which eventually increases yield and juiciness of meat. Moreover, the phosphate salts used for the preparation of marinades are not considered as antimicrobials. Therefore, more attention is paid to influence of different phosphates and their combinations with organic acid salts and other ingredients on the microbiological status and quality of meat. Chicken meat, compared to other types of meat, has many advantages. However, chicken meat is one of the most commonly identified source of Salmonella outbreaks in humans and cause of poisoning commonly referred to Salmonella Enteritidis and Salmonella Typhimurium. Moreover, the only legislation defined safety criteria for semi- processed meat, which is the marinated chicken meat, is the absence of Salmonella spp. Therefore, it is scientifically and professionally justified to investigate the influence of marination on the growth and survival of Salmonella species in broiler meat, as it is conducted in this study. Furthermore, research in the framework of this dissertation, was aimed to demonstrate the influence of marination on microbiological indicators of hygienic status and shelf- life of meat, as well as the chemical and physical and chemical parameters of broiler meat quality...

Sposobnost da kontaminira hranu pripremljenu za konzum, visoko prerađene proizvode i proizvode skladištene na niskim temperaturama kao i relativno visok stepen smrtnosti i izrazita sosobnost adaptacije na uslove okoline svrstavaju Listeria monocytogenes u grupu patogenih mikroorganizama prenosivih hranom koji predstavljaju jedan od najvećih izazova u oblasti javnog zdravlja. Adaptacija L. monocytogenes na uslove sredine i sposobnost dugotrajnog opstanka u objektima za proizvodnju i preradu hrane ključni su razlozi za kontaminaciju različitih proizvoda. Poslednjih godina, višestruka rezistencija bakterija na različita antimikrobna sredstva nametnula se kao jedna od najznačajnijih tema u oblasti zdravstva. Izlaganje brojnim antimikrobnim sredstvima (antibiotici, dezinficijensi kao i sveprisutni teški metali) omogucava selektivni pritisak za razvoj rezistencije uz propratnu unakrsnu rezistenciju i doprinosi povećanju vitalnosti bakterija u sredini u kojoj se nalaze. U ovom radu prikazani su neki od mogućih mehanizama adaptacije koji mogu doprineti i doprinose opstanku L. monocytogenes u sredini kao i sposobnost rasta ovog patogena u različitim tipovima hrane. Prikaz uključuje i mehanizme adaptacije na teške metale kao što je kadmijum, dezinficijense kao bezalkonijum hlorid (BC), odabrane antibiotike (fluorokvinolon ciprofloksacin, na primer) i druge toksične supstance (tetrafenil-fosfonijum, koji se često koristi kao anti-kancerogeno sredstvo ali i substrat efluks pumpe). U cilju karakterizacije uticaja višestruke rezistencije na preživljavanje i rast L. monocytogenes u razlicitim tipovima hrane, testirali smo mutante adaptirane na visoke koncentracije antibiotika ciprofloksacina i dezinficijensa bezalkonijum hlorida. Ispitivanjem kolekcije mutanata roditeljskog soja L. monocytogenes 10403S sa umetnutim mariner-transpozonom, registrovali smo mutante sa karakteristikama tetR mutanta sa povecanom rezistencijom na tetrafenil-fosfonijum. Konacno, testirali smo i odabrane mutante sa transpozonom koji su u toku eksperimenta iskazali promene u fenotipu nastale kao posledica odgovora organizama na razlicite uslove stresa (osetljivost na niske temperature, ne-hemoliticne sojeve, sojeve koje pripadaju epidemskom klonu II a osetljive na dejstvo faga ), mutante nastale kao posledica genetskog brisanja proteina wap i genetskog regiona 18, kao i odabrane kliničke izolate i izolate iz hrane... Ability to contaminate ready-to-eat, highly processed, cold-stored food products, relatively high fatality rate and environmental adaptations define .Listeria monocytogenes as a challenging foodborne pathogen of high public health significance. Listeria’s adaptations and prolonged persistence in food processing facilities are a key link to contamination of foods. In recent years, bacterial multidrug resistance has been recognized as an emerging health challenge. Exposure to antimicrobials (antibiotics, disinfectants or environmentally ubiquitous heavy metals), may provide selective pressures for resistance with accompanying cross-resistance and increase in environmental fitness. In this work I addressed some of the possible mechanisms of adaptations that may contribute to persistence of L. monocytogenes in the environment as well as to survival and growth in different foods. This includes adaptations to heavy metals such as cadmium, disinfectants such as benzalkonium chloride (BC), selected antibiotics (e.g. the fluoroquinolone ciprofloxacin) and other toxic compounds (e.g. tetraphenyl-phosponium, often used as an anti-carcinogenic and efflux pump substrate). In order to characterize the impact of multidrug resistance on survival and growth of L. monocytogenes in different foods, we tested mutants adapted to higher concentrations of antibiotic ciprofloxacin and disinfectant benzalkonium chloride. By screening of the mariner-based mutant librarie of L monocytogenes 10403S we detected tetR-like mutants with increase in the resistance to tetraphenyl-phosponium. Lastly, we tested selected mariner-based mutants with the change in the response to number of different stresses (cold sensitive, non-hemolytic, resistance to phage in epidemic clone II strains) deletion mutants of wap and region18, as well as selected food and clinical isolates. Selection of several L. monocytogenes strains on either ciprofloxacin (2 μg/ml) or the BC(10 μg/ml) led to derivatives with increased MICs not only to these agents but also to several other compounds, including the antibiotic gentamicin, the dye ethidium bromide, and the chemotherapeutic drug tetraphenyl - phosphonium chloride. The spectrum of compounds to which these derivatives exhibited reduced susceptibility was the same regardless of whether they were selected on ciprofloxacin or on BC...

U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije. Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima, radeno je metodom najverovatnijeg moguceg broja (MPN). Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a, utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pcela 110/kg. U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum. Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih metoda mikrobiologije. Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda. U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne rasporedenosti spora u uzorcima. In this study, the presence of Clostridium botulinum spores in different types of honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples was included in this investigation and samples originated from different regions of the Republic of Serbia. In addition to classical microbiological methods, after the dilution, enrichment, spinning and membrane filtration, PCR and multiplex PCR methods were used. Determination the number of spores in the PCR positive samples, was done using the most likely possible number (MPN) methodology. According to the results obtained by conventional microbiological methods and multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg. In one honey sample, spores of type B C. botulinum were detected, in one honey sample spores of type E, in two honey samples spores of types A and E, and in one sample spores of three types of C. botulinum (A, B, E) were detected. Type E Clostridium botulinum spores were detected in a sample of honey bees. In the same samples of honey or honey bees, using conventional qualitative and quantitative microbiological methods, spores of Clostridium botulinum were not detected. The detection of Clostridium botulinum spores with PCR, multiplex PCR and conventional microbiological methods in honey and honey bee samples is not possible without preenrichment. Conventional microbiological methods, including SRPS ISO 15213:2011, are not suitable for the detection of Clostridium botulinum spores in honey samples. Due to the low sensitivity of these methods, they can lead to false negative results. In the honey samples which served as a positive control and wich were contaminated with Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of one spore/g of honey. In order to detect Clostridium botulinum spores in honey and honey bees samples using PCR method, due to the small and/or unequal distribution of spores in the samples, the test had to be repeated at least three times for each sample.

In this study, the presence of Clostridium botulinum spores in different types of honey and honey bees was examined. The sum of 59 honey and 61 honey bee samples was included in this investigation and samples originated from different regions of the Republic of Serbia. In addition to classical microbiological methods, after the dilution, enrichment, spinning and membrane filtration, PCR and multiplex PCR methods were used. Determination the number of spores in the PCR positive samples, was done using the most likely possible number (MPN) methodology. According to the results obtained by conventional microbiological methods and multiplex PCR, the presence of Clostridium botulinum spores, was detected in 5 honey samples (8.47%) and 1 (1.64%) honey bee sample. Number of spores in positive honey samples was from 20/kg to 204/kg, and in a sample of honey bees was 110/kg. In one honey sample, spores of type B C. botulinum were detected, in one honey sample spores of type E, in two honey samples spores of types A and E, and in one sample spores of three types of C. botulinum (A, B, E) were detected. Type E Clostridium botulinum spores were detected in a sample of honey bees. In the same samples of honey or honey bees, using conventional qualitative and quantitative microbiological methods, spores of Clostridium botulinum were not detected. The detection of Clostridium botulinum spores with PCR, multiplex PCR and conventional microbiological methods in honey and honey bee samples is not possible without preenrichment. Conventional microbiological methods, including SRPS ISO 15213:2011, are not suitable for the detection of Clostridium botulinum spores in honey samples. Due to the low sensitivity of these methods, they can lead to false negative results. In the honey samples which served as a positive control and wich were contaminated with Clostridium botulinum NCTC 7272 strain, the PCR method may prove a presence of one spore/g of honey. In order to detect Clostridium botulinum spores in honey and honey bees samples using PCR method, due to the small and/or unequal distribution of spores in the samples, the test had to be repeated at least three times for each sample. U ovom radu vršeno je ispitivanje prisustva spora Clostridium botulinum u uzorcima razlicitih vrsta meda i medonosnih pcela. Ispitano je ukupno 59 uzoraka meda i 61 uzorak medonosnih pcela, poreklom iz razlicitih regona Republike Srbije. Pored klasicnih mikrobioloških metoda u laboratorijskim ispitivanjima je posle postupka dilucije, predobogacenja, centrifugovanja i membranske filtracije primenjena metoda multipleks PCR i PCR. Odredivanje broja spora, u PCR pozitivnim uzorcima, radeno je metodom najverovatnijeg moguceg broja (MPN). Prisustvo spora Clostridium botulinum, metodom multipleks PCR i PCR-a, utvrdeno je u 5 uzoraka (8,47%) meda i jednom (1,64%) uzorku medonosnih pcela. Broj spora u pozitivnim uzorcima meda bio je od 20/kg do 204/kg, a u uzorku medonosnih pcela 110/kg. U jednom uzorku meda detektovane su spore B tipa C. botulinum, u jednom uzorku je detektovan E tip, u dva uzorka su detektovane spore istovremeno dva tipa C.botulinum (A, E) i u jednom uzorku istovremeno tri tipa C. botulinum (A, B, E). Spore Clostridium botulinum tipa E dokazane su u jednom uzorku medonosnih pcela. U istim uzorcima meda, odnosno medonosnih pcela, klasicnim kvalitativno-kvantitativnim metodama nije dokazano prisustvo spora Clostridium botulinum. Detekcija spora Clostridium botulinum direktno iz neobradenih uzoraka meda i medonosnih pcela, bez predobogacenja, nije moguca primenom metoda PCR i klasicnih metoda mikrobiologije. Klasicne metode mikrobiologije, ukljucujuci i metodu SRPS ISO 15213:2011, nisu podesne za detekciju spora Clostridium botulinum u uzorcima meda. Zbog niske osetljivosti navedene metode daju lažno negativne rezultate. U uzorku meda koji je služio kao pozitivna kontrola i koji je prethodno namerno kontaminiran referentnim sojem Clostridium botulinum NCTC 7272, PCR metodom može da se dokaže jedna spora/g meda. U cilju detekcije spora Clostridium botulinum u uzorcima meda i medonosnih pcela primenom metode PCR, za svaki ispitujuci uzorak moraju se izvoditi višestruka ponovljanja, ispitivanja jednog istog uzorka, zbog malog broja i/ili neravnomerne rasporedenosti spora u uzorcima.

Cilj ove doktorske disertacije bio je ispitivanje kinetike izolacije ekstrakata sa jakim antibakterijskim delovanjem iz biljnog materijala primenom različitih postupaka ekstrakcije. Za izolaciju bioaktivnih supstanci korišćena je natkritična ekstrakcija sa ugljenik(IV)-oksidom, hidrodestilacija i ekstrakcija etanolom sa i bez primene ultrazvuka. Predmetom istraživanja ove doktorske disertacije obuhvaćena je analiza i optimizacija različitih procesa izolacije, kao i ispitivanje sadržaja aktivnih komponenti i/ili antibakterijskog dejstva dobijenih ekstrakta za primenu u farmaceutskoj i/ili prehrambenoj industriji. U eksperimentima je, kao sirovina, korišćeno odabrano začinsko i lekovito bilje (Rosmarinus officinalis, Salvia officinalis, Thymus vulgaris, Origanum vulgare, Eugenia caryophyllata, Laurus nobilis, Angelica archangelica, Echinacea angustifolia, Verbascum thapsus, Calendula officinalis i Aloe barbadensis Miller), lišaj Usnea barbata i odabrane mešavine biljnog materijala (karanfilić/origano i karanfilić/timijan). U disertaciji je posebno ispitana i analizirana kinetika procesa natkritične ekstrakcije. S tim u vezi, ispitivan je uticaj operativnih parametara (pritiska i temperature) i odgovarajućeg predtretmana biljnog materijala sa natkritičnim ugljenik(IV)-oksidom na kinetiku natkritične ekstrakcije, kao i uticaj prisustva pojedinih biljnih sirovina u smeši (njihovih glavnih komponenata) na povećanje moći rastvaranja natkritičnog ugljenik(IV)- oksida (kosolventski efekat), pa samim tim i na povećanje brzine ekstrakcije i smanjenje potrošnje natkritičnog fluida. U disertaciji su, takođe, prikazani rezultati ispitivanja kinetike ekstrakcije etanolom sa i bez primene ultrazvuka. Za simulaciju procesa natkritične ekstrakcije iz pojedinačnih biljnih sirovina i izabranih mešavina biljnog materijala korišćeni su postojeći matematički modeli zasnovani na diferencijalnom bilansu mase (Stamenic i sar., 2008; Zizovic i sar., 2005; Sovova i sar., 1994a) i na analogiji sa prenosom toplote (Bartle i sar., 1990; Hong i sar., 1990; Reverchon i sar., 1994 i 1993). Odgovarajući parametri dobijeni primenom navedenih matematičkih modela, zajedno sa podacima o hemijskom sastavu, korišćeni su za interpretaciju fenomena prenosa mase u čvrstoj fazi i u filmu natkritičnog ugljenik(IV)-oksida do kojih dolazi tokom procesa natkritične ekstrakcije iz pojedinih biljnih sirovina i izabranih mešavina biljnog materijala. Za modelovanje procesa ekstrakcija sa etanolom korišćeni su postojeći modeli izvedeni iz drugog Fikovog zakona u cilju određivanja koeficijenata difuzije ekstraktivnih materija u čvrstoj fazi (Crank, 1975; Treybal, 1968). Za rešavanje jednačina navedenih matematičkih modela korišćen je softverski paket Microsoft Fortran PowerStation 4.0. Za ispitivanje antibakterijskog delovanja biljnih ekstrakata i etarskih ulja korišćenа је modifikovanа mikrodiluciona ili makrodiluciona metoda u bujonu opisana i preporučena od strane CLSI (Clinical Laboratory Standards Institute, 2008, SAD), koja se u rutinskoj laboratorijskoj praksi koristi za ispitivanje antibakterijskog delovanja antibiotika, tj. za određivanje vrednosti MIC (minimalne inhibitorne koncentracije) antibiotika. Antibakterijsko delovanje biljnih ekstrakata ispitivano је na sojevima: Staphylococcus vrsta, uključujući sojeve Staphylococcus aureus koji su rezistentni na meticilin (MRSA), Enterococcus vrsta (uključujući vankomicin rezistentne enterokoke, VRE), Streptococcus, Bacillus i Geobacillus vrsta. Ispitan je uticaj pojedinih ekstrakta i na sojeve Gram-negativnih bakterija, Е.coli, Enterobacter cloacae i Sallmonela Enteritidis... This study was aimed to investigate kinetics of isolation of the extracts with strong antibacterial activity from plant material by using different extraction processes. The bioactive substances from plant matrial were isolated by supercritical carbon dioxide extraction, hydrodistillation and ethanol extraction with and without ultrasound. The scope of the present study was kinetic analysis and optimization of different extraction processes as well as content of active substance and/or antibacterial activity of isolated supercritical extracts for application in pharmaceutical and/or food industry. Selected herbs and spices (Rosmarinus officinalis, Salvia officinalis, Thymus vulgaris, Origanum vulgare, Eugenia caryophyllata, Laurus nobilis, Angelica archangelic), Echinacea angustifolia, Verbascum thapsu, Calendula officinalis, Aloe barbadensis Miller), lichen Usnea barbata and selected mixtures of plant materials (clove/oregano and clove/thymus) were used as raw material for experiments. In this study, supercritical carbon dioxide extraction kinetics was particularly analyzed. Therewith, influence of operating parameters (pressure and temperature) and pretreatment of the plant material with supercritical carbon dioxide on the supercritical extraction kinetics, influence of presence of particular plant material in the mixtures of plants (their main components) on the solubility power of supercritical carbon dioxide (co-solvent effect) and subsquently on increase of extraction rate and reduction of supercritical fluid consumption was investigated in this work as well. The results of investigation of the kinetics of ethanol extractions are also presented here. Mathematical models based on differential mass balance (Stamenic et al., 2008; Zizovic et al., 2005; Sovova et al., 1994a,b) and heat transfer analogy (Bartle et al., 1990; Hong et al., 1990; Reverchon et al., 1994, 1993) were used for simulation of process of supercritical carbon dioxide extraction from pure plants and chosen mixture of plants for evaluation of parameters that can be used for interpretation of the mass transfer phenomena in the solid and supercritical carbon dioxide phase. Mathematical models based on the Second Fick’s Law were used for simulation of ethanol extractions in order to evaluate coefficients of diffusion of extractible substances in solid phase (Crank, 1975; Treybal, 1968). Solution of the equations of above metioned mathematical models were solved using Microsoft Fortran PowerStation 4.0. For antibacterial susceptibility testing, modified broth microdilution оr macrodilution method was applied in accordance with the prescription of CLSI (Clinical Laboratory Standards Institute, 2008, USA and corresponding MIC (minimal inhibitory concentration) values were determined. Antibacterial effect of plant extracts on Staphylococcus species including meticillin-resistant Staphylococcus aureus (MRSA) bacterial strains as well as Enterococcus (including vancomycin resistant strains–VRE), Streptococcus, Bacillus and Geobacillus species was investigated. Antibacterial activity of extracts on the Gram-negative bacteria such as Е.coli, Enterobacter cloacae and Sallmonela Enteritidis was investigated as well...