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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 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.

Izolacija i identifikacija Salmonella spp. ostaje nezamenjiva kao potvrdna metoda u mikrobiologiji hrane i hrane za životinje. Međutim, savremena industrija hrane, ali i javno zdravstvo zahtevaju razvoj novih, brzih metoda za detekciju Salmonella spp., jednog od najvažnijih patogenih mikroorganizama prenosivih hranom. Pored toga, tokom epidemioloških studija, uglavnom nije dovoljno da se izolati Salmonella tipiziraju do nivoa vrste i serotipa, već je neophodna i primena metoda tipizacije koje mogu napraviti razliku između epidemiološki različitih, ali genetski srodnih izolata. Veliki broj alternativnih metoda, uključujući i real-time PCR protokole, razvijen je i validovan u cilju detekcije Salmonella spp. Pored toga, napredak u razvoju molekularnih metoda pružio je alate visoke diskriminatorne moći koji su posebno mesto primene zauzeli pri određivanju izvora kontaminacije hrane, omogućavajući brzu i izuzetno pouzdanu tipizaciju Salmonella spp. Ova doktorska disertacija obuhvata četiri osnovne celine: (1)“In house” validacija real-time PCR 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 protokola za detekciju invA gena Salmonella spp. u hrani; (4) Molekularna karakterizacija i ispitivanje antimikrobne 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 Salmonella Enteritidis i 60 izolata S. Typhimurium (po 12 poreklom ljudi, 24 poreklom iz hrane i 24 poreklom iz fecesa živine), a zatim je izvršena njihova molekularna karakterizacija i ispitivanje antimikrobne osetljivosti. Ispitivanje antimikrobne osetljivosti Salmonella izvršeno je disk difuzionom metodom prema EUCAST protokolu, dok je za određivanje minimalne inhibitorne koncentracije pri, method in the microbiology of food and feed. However, the modern food industry and public health require the development of new, rapid methods for the detection of Salmonella spp., one of the most important foodborne pathogens. In addition, during epidemiological studies, it is generally 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 between epidemiologically distinct, but genetically related isolates. A number of alternative methods, including real-time PCR protocols, have been developed and validated for the detection of Salmonella spp. In addition, the advances in the development of molecular methods have provided tools of high discriminatory power that have been particularly useful in determining food 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 protocol for the detection of invA and ttr genes of Salmonella spp. in food and feed; (2) In-house validation 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 protocol for the detection of the invA gene of Salmonella spp. in food; (4) Molecular characterization and antimicrobial susceptibility testing of Salmonella Enteritidis and S. Typhimurium isolated from food, feed and feces. An extensive validation study, where real-time PCR protocols for the detection of invA and ttr of Salmonella spp. in food and feed were tested, gave excellent results and showed that they can be used as an adequate replacement for the standard method. By comparing different DNA extraction procedures, lower Ct values were achieved by using "Chelex extraction" rather than by extraction based on the thermal lysis of the cell...

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.

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.

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.