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Cilj istraživanja u okviru ove doktorske disertacije bio je utvrđivanje ukupne prevalencije Y. enterocolitica kod svinja i karakterizacija izolata u odnosu na bioserotip, prisustvo gena virulencije, genetski diverzitet i otpornost na antimikrobne lekove. Proučavani su mogući faktori rizika u proizvodnji svinja povezani sa infekcijom Y. enterocolitica koji nastaju u fazama pre klanja i za vreme klanja. Tokom četiri sezone pregledano je ukupno 960 uzoraka tonzila svinja poreklom sa farmi otvorenog i zatvorenog tipa zaklanih na tri različite klanice. Svinje su bile poreklom sa ukupno 30 različitih farmi raspoređenih u pet regiona u Republici Srbiji. Prilikom svakog uzorkovanja popunjavan je upitnik o uslovima na farmi kao i uslovima u toku transporta i vremenu boravka u depou klanice. Ukupna prevalencija Y. enterocolitica kod svinja iznosila je 10,4%. Najčešći bioserotip Y. enterocolitica bio je BT4/O:3 (81,6%). Patogenost 63 izolata Y. enterocolitica BT4/O:3 (uključujući i BT4 S-/O:3), potvrđena je prisustvom gena virulencije ail i ystA i odsustvom gena ystB (100%). Karakterizacijom 63 potvrđena izolata pomoću PFGE identifikovano je pet različitih genotipova sa zajedničkim identičnim genetskim profilima (100% sličnosti) unutar svakog genotipa. Četiri genotipa potiču sa oba tipa farmi i sa tri različite klanice, što ukazuje na visoku genetsku stabilnost i prostornu cirkulaciju patogenog mikroorganizma. Disk difuzionom metodom utvrđeno je da su svi izolati Y. enterocolitica bili su osetljivi na cefotaksim, ceftazidim, meropenem, gentamicin, trimetroprim i sulfonamide. Izolati koji potiču sa zatvorenog tipa farmi bili su rezistentni samo na ampicilin, dok je rezistencija na ampicilin, nalidiksinsku kiselinu, tetraciklin i hloramfenikol utvrđena na farmama otvorenog tipa. Metodom multivarijantne logističke regresije utvrđeni su faktori rizika koji zajedno doprinose pojavi infekcije svinja sa Y. enterocolitica i uključuju farme otvorenog tipa (odnos verovatnoće, OR=2,31, 9, The aim of the research within this doctoral dissertation was to determine prevalence of Y. enterocolitica in pigs of slaughter age and to characterize the isolates in relation to bio-serotype, the presence of virulence genes, genetic diversity, and antimicrobial resistance. Moreover, possible risk factors associated with Y. enterocolitica infection during the preharvested and harvested phase of pig production were studied. During four seasons, a total of 960 tonsil samples from pigs originating from farow to finish and fattening farms slaughtered at three different slaughterhouses were examined. The pigs came from a total of 30 different farms distributed in five regions in Serbia. During each sampling, a questionnaire was filled out about the conditions on the farm as well as the conditions during transport and the time of stay in the lairage. The overall Y. enterocolitica prevalence in the pigs was 10.4%. The most common Y. enterocolitica bioserotype was 4/O:3, accounting for 81.6% of investigated isolates. The pathogenicity of 63 Y. enterocolitica 4/O:3 (including 4S-/O:3) isolates, originating from all infected farms, was confirmed by the presence of both the ail and ystA virulence-associated genes and the absence of ystB gene (100%). Characterization with PFGE of 63 confirmed Y. enterocolitica isolates identified five different genotypes with shared identical genetic profiles (100% similarity) within each genotype. The four genotypes originate from both types of farms and from three different slaughterhouses, which indicates high genetic stability and spatial circulation of the pathogen. Using the disk diffusion method, it was determined that all isolates of Y. enterocolitica were susceptible to cefotaxime, ceftazidime, meropenem, gentamicin, trimethoprim and sulfonamides. Isolates originating from farrow-to-finish farms were only resistant to ampicillin, while resistance to nalidixic acid, tetracycline, and chloramphenicol at fattening farms was also observed

West Nile virus (WNV) can affect humans, birds, horses and another mammals, causing asymptomatic infection, mild febrile disease, neurological and systematic disease and death. In order to gain insight into the prevalence of WNV, a monitoring program has been established in the Republic of Serbia. Whole genome sequencing is essential for the molecular epizootiological analysis of virus entry and transmission routes, especially in high-risk regions. This paper describes the development of a multiplex PCR based NGS protocol for whole genome sequencing of WNV lineage 2 directly from biological samples using Oxford Nanopore (ONT) platform. The results obtained using this platform, confirmed by Sanger sequencing, indicate that this protocol can be applied to obtain whole sequences of the WNV genome, even when the virus concentration in the sample is medium, Ct value is approximately 30. The use of this protocol does not require prior virus isolation on cell culture nor the depletion of host nucleic acids.

Yersiniosis caused by Yersinia enterocolitica is the third most common zoonosis transmitted from asymptomatic, healthy pigs to humans through raw or insufficiently cooked meat. The occurrence of Y. enterocolitica on a farm can vary, depending on different risk factors, including production system and biosecurity level. At the slaughterhouse, the contamination level of carcasses depends to a great extent on practices during lairage, along with the handling and processing of the head, tongue, tonsils, and rectum of slaughtered pigs. A comprehensive approach for further Y. enterocolitica farm/slaughterhouse categorization, improved hygiene practices, and mandatory surveillance for underestimated pathogens within the food chain is necessary for maintaining the One Health concept.

The aim of this research was to examine the chemical properties of freshly squeezed wild garlic extract (FSWGE) and its use as an additive in burgers (BU). Technological and sensory properties of such fortified burgers (BU) were determined. LC-MS/MS analyses identified thirty-eight volatile BAC. Allicin prevalence (11.375 mg/mL) is the key parameter determining the amount of FSWGE added in raw BU (PS-I 1.32 mL/kg, PS-II 4.40 mL/kg, and PS-III 8.79 mL/kg). Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) values of the FSWGE and evaporated FSWGE (EWGE) were determined against the six microorganisms using a microdilution method. The data indicated that using FSWGE can result in a reduced risk of Serratia marcescens (MIC = 50 mg/mL; MBC = 60 mg/mL), Listeria monocytogenes (MIC = MBC = 90 mg/mL), Escherichia coli and Staphylococcus aureus (MIC = 90 mg/mL; MBC ≥ 100 mg/mL), and Salmonella enteritidis and Enterococcus faecium (MIC = 100 mg/mL; MBC > 100 mg/mL) in BU. Changes in antioxidant (AOX) capacity were followed during cold storage (up to 10 days) and freezing (90 days). It was shown that PS-III had the highest level of AOX capacity during the entire period of cold storage, revealing 8.79 mL FSWGE/kg BU as the most suitable effective concentration. Adding FSWGE did not negatively affect the technological and physico-chemical properties during both cold and freeze storage. Regarding sensory evaluation, modified BU received mostly higher scores compared to control. The results of this study have demonstrated the great potential of wild garlic extract usage in the creation of safe products with prolonged shelf life. © 2023 by the authors.

A survey was undertaken to determine the overall prevalence of Yersinia enterocolitica in pigs of slaughter age and to characterize the isolates in relation to bio-serotype, the presence of virulence genes, genetic diversity, and antimicrobial resistance. Moreover, possible risk factors associated with Y. enterocolitica infection during the pre-harvested and harvested phase of pig production were studied. The overall Y. enterocolitica prevalence in the pigs was 10.4% (95% confidence interval, CI = 8.5-12.3%). The most common Y. enterocolitica bio-serotype was 4/ O:3, accounting for 81.6% of investigated isolates. The pathogenicity of 63 Y. enterocolitica 4/O:3 isolates, originating from all infected farms, was confirmed by the presence of both the ail and ystA virulence-associated genes and the absence of ystB gene (100%). Characterization with PFGE of 63 confirmed Y. enterocolitica 4/O:3 isolates identified five different genotypes with shared identical genetic profiles (100% similarity) within each genotype. Isolates originating from farrow-to-finish farms were only resistant to ampicillin, while resistance to nalidixic acid, tetracycline, and chloramphenicol at fattening farms was also observed. Risk factors related to Y. enterocolitica pig infection include fattening farms (odds ratio, OR = 2.3, 95% CI= 1.4-3.8, P LT 0.001), a 3-6 h lairage period (OR = 1.6, 95% CI = 1.0-2.6, P = 0.035) and winter season (OR = 3.8, 95% CI = 2.0-7.4, P LT 0.001). In addition to the overall characterization of Y. enterocolitica isolates, identification of the main risks associated with infection allows better application of preventive measures to reduce the occurrence and distri-bution of Y. enterocolitica infection.

Izolacija i identifikacija Salmonella spp. ostaje nezamenjiva kao potvrdna metoda umikrobiologiji hrane i hrane za životinje. Međutim, savremena industrija hrane, ali i javnozdravstvo zahtevaju razvoj novih, brzih metoda za detekciju Salmonella spp., jednog odnajvažnijih patogenih mikroorganizama prenosivih hranom. Pored toga, tokomepidemioloških studija, uglavnom nije dovoljno da se izolati Salmonella tipiziraju do nivoavrste i serotipa, već je neophodna i primena metoda tipizacije koje mogu napraviti razlikuizmeđu epidemiološki različitih, ali genetski srodnih izolata. Veliki broj alternativnihmetoda, uključujući i real-time PCR protokole, razvijen je i validovan u cilju detekcijeSalmonella spp. Pored toga, napredak u razvoju molekularnih metoda pružio je alate visokediskriminatorne moći koji su posebno mesto primene zauzeli pri određivanju izvorakontaminacije hrane, omogućavajući brzu i izuzetno pouzdanu tipizaciju Salmonella spp.Ova doktorska disertacija obuhvata četiri osnovne celine: (1)“In house” validacija real-timePCR protokola za detekciju invA i ttr gena Salmonella spp. u hrani i hrani za životinje; (2)„In house“ validacija real-time PCR protokola za detekciju Salmonella Enteritidis i S.Typhimurium u hrani; (3) Razvoj i „in house“ validacija novog real-time PCR protokolaza detekciju invA gena Salmonella spp. u hrani; (4) Molekularna karakterizacija i ispitivanjeantimikrobne osetljivosti Salmonella Enteritidis and S. Typhimurium izolovanih iz hrane,hrane za životinje i fecesa.Tokom izrade ove doktorske disertacije formirana je kolekcija od 60 izolata SalmonellaEnteritidis i 60 izolata S. Typhimurium (po 12 poreklom ljudi, 24 poreklom iz hrane i 24poreklom iz fecesa živine), a zatim je izvršena njihova molekularna karakterizacija iispitivanje antimikrobne osetljivosti. Ispitivanje antimikrobne osetljivosti Salmonellaizvršeno je disk difuzionom metodom prema EUCAST protokolu, dok je za određivanjeminimalne inhibitorne koncentracije primenjen E test. Molekula, method in the microbiology of food and feed. However, the modern food industry and publichealth require the development of new, rapid methods for the detection of Salmonella spp., oneof the most important foodborne pathogens. In addition, during epidemiological studies, it isgenerally not sufficient to determine Salmonella isolates up to the species or the serotype level,but it is also necessary to apply subtyping methods that can distinguish betweenepidemiologically distinct, but genetically related isolates. A number of alternative methods,including real-time PCR protocols, have been developed and validated for the detection ofSalmonella spp. In addition, the advances in the development of molecular methods haveprovided tools of high discriminatory power that have been particularly useful in determiningfood contamination sources, enabling fast and highly reliable typing of Salmonella spp.This dissertation has four major sections: (1) In-house validation of the real-time PCR protocolfor the detection of invA and ttr genes of Salmonella spp. in food and feed; (2) In-housevalidation of the real-time PCR protocol for the detection of Salmonella Enteritidis and S.Typhimurium in food; (3) Development and in-house validation of new real-time PCR protocolfor the detection of the invA gene of Salmonella spp. in food; (4) Molecular characterizationand antimicrobial susceptibility testing of Salmonella Enteritidis and S. Typhimurium isolatedfrom food, feed and feces.An extensive validation study, where real-time PCR protocols for the detection of invA and ttrof Salmonella spp. in food and feed were tested, gave excellent results and showed that theycan be used as an adequate replacement for the standard method. By comparing different DNAextraction procedures, lower Ct values were achieved by using "Chelex extraction" rather thanby extraction based on the thermal lysis of the cell...

A total of 61 honey bees from different regions of the Republic of Serbia were analyzed for Clostridium botulinum (C. botulinum) spores. The microbiological methods and molecular methods (multiplex PCR/mPCR and PCR method) were utilized to examine multiple subunits of each honey bees samples. The C. botulinum spores in PCR-positive samples were estimated by the most probable number method (MPN). The presence of C. botulinum spores, by applying mPCR and PCR methods, was detected in 1 of the 61 honey bees (1.64%). Using MPN method, the number of spores in positive sample was 110/kg. Detection of C. botulinum spores directly from untreated honey bees, without prior enrichment, is impossible by applying PCR. Using conventional microbiological methods, detection of C. botulinum spores in dead honey bees is not possible without preenrichment. Therefore, conventional, microbiological methods are not suitable for the detection of C. botulinum spores in honey bees. In order to detect C. botulinum spores in honey bees using PCR methods, due to the small and/or unequal distribution of spores in the samples, it is desirable to use multiple subunits/replicates for each sample examined.

The aim of this paper was to evaluate two real-time PCR (qPCR) protocols for the detection of Salmonella spp. in minced meat and chicken neck skin, after DNA extraction using the InstaTM Gene matrix (BioRad, USA) and DNA extraction based on thermal cell lysis. The applied molecular methods were sensitive and specific for the rapid detection of Salmonella spp. in minced meat and chicken neck skin. The qualitative results were identical regardless of the applied DNA extraction or qPCR protocols. Lower Cq values were achieved after DNA extraction using the InstaTM Gene matrix.

After a human trichinellosis outbreak in Zlatibor District, Serbia, in 2016, Trichinella larvae were found in wild boar (Sus scrofa) meat products. One hundred and fourteen people were infected during the outbreak. The larvae were determined to be Trichinella britovi using the polymerase chain reaction method. Trichinella britovi has previously been identified in Serbia, but this is the first case of the species being confirmed in food samples linked to human trichinellosis. The results of the study confirmed that the T. britovi is able to affect human health. In addition, this study suggests the role of wild boars as reservoirs of T. britovi in Serbia.

The microbiological standard method for the detection of Salmonella spp. is time-consuming, and consequently there is a need for an alternative rapid methodology for its detection. In this study, two open-formula real-time PCR methods for detecting the inv A and ttr gene Salmonella spp. have been successfully optimized and in-house validated. Different DNA extraction procedures were used (boiling, Chelex resin and standard I - Biorad). The relative sensitivity and false positive ratio for the alternative methods were 100% and 0.0%, respectively. The relative trueness was in range from 96.8% to 99.2%. No false negative results were detected. The lowest C-t values were obtained using the protocol for detecting the ttr gene after DNA extraction by the Chelex. The results were compared in a large number of food and environmental samples to those of the SRPS EN ISO 6579:2008 standard method and commercial kit (IQ check((R))Salmonella II kit, Bio-Rad, USA). Practical applicationsThe whole procedure of real-time PCR methods in this study was approximately 20 hr, in contrast to 4-5 days of analysis time for the standard method (SRPS EN ISO 6579:2008). The real-time PCR method also provides a high level of sensitivity and specificity with a low risk of cross-contamination. The implementation of the method for routine analysis will help improve safety in the food production chain, by providing results compatible with the ISO standard, but more rapidly.

Wild animals, including red foxes (Vulpes vulpes) and golden jackals (Canis aureus), are the most important reservoirs of Trichinella spp. Although the red fox is considered one of the main reservoirs of Trichinella spp. in Europe, only a few animals have been examined in Serbia. The present study assessed Trichinella spp. infection in red foxes and golden jackals from the six districts in Serbia. Thirty-seven carcasses of red foxes and 13 carcasses of golden jackals shot during the official hunting season were examined. Larvae of Trichinella spp. were detected in 13 (35%) of 37 red foxes and in 8 (61%) of 13 golden jackals. Trichinella spiralis and Trichinella britovi were the only two species identified after a multiplex polymerase chain reaction analysis. Trichinella britovi infection was detected in 85% of red foxes and in 38% of golden jackals, and T. spiralis was detected in 15% of red foxes and in 63% of golden jackals. The findings emphasize the need for an active surveillance program for Trichinella spp. infection in wildlife in Serbia and the whole of the Balkans, with special attention on the red fox because it is widespread and occurs in high densities.

Introduction: The aim of this study was to determine the prevalence of Salmonella along the slaughter line and to identify possible critical control points in one slaughterhouse facility located in the city of Belgrade. Methodology: In total, 700 samples were tested: two swabs from both sides of carcass were taken from each of 100 pigs. In this way, 200 pig skin swab samples were taken after stunning, 200 after processing and 200 after chilling. Additional 100 samples of ileal contents were also taken from the same pigs to obtain a collection of 270 isolates. All samples were analyzed using standard culture methods and serotyping. PFGE was performed for 27 isolates. Determination of antimicrobial resistance was performed by E-test. Results: In total, 47 (23.5%) swab samples were positive for the presence of Salmonella after stunning. After processing, Salmonella was isolated in two swab samples (1%), whereas all samples which were collected after chilling were negative for Salmonella. The sampling of ileal contents was positive for five Salmonella isolates (5%). The most frequently isolated serotypes were S. Derby (90.74%), S. Infantis (5.56%) and S. Typhimurium (3.7%). All tested isolates were resistant to tetracycline. Resistance was recorded to nalidixic acid (23.3%), ciprofloxacin (20%), ampicillin (10%) and chloramphenicol (14.4%), as well. The PFGE results indicated that isolates had a high genetic similarity. Conclusions: The investigation has confirmed that bacteriological examinations of carcass swabs, as well as ileal content, could be used to assess the carriage of salmonellae in pigs at the time of slaughter.