TRICHINELLA INFECTIONS IN RED FOXES (VULPES VULPES) AND GOLDEN JACKALS (CANIS AUREUS) IN SIX DISTRICTS OF SERBIA.

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.

Techno-Functional Properties of Burgers Fortified by Wild Garlic Extract: A Reconsideration.

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.

Development of multiplex PCR based NGS protocol for whole genome sequencing of West Nile virus lineage 2 directly from biological samples using Oxford Nanopore platform.

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.

Risk factors and the overall characterization of Yersinia enterocolitica as an initial model of pathogen surveillance in the pig production system in Serbia.

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 < 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 < 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 distribution of Y. enterocolitica infection.

Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004-2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Trichinella britovi in Game Meat Linked to Human Trichinellosis Outbreak in Serbia.

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.

Prevalence of Salmonella enterica in slaughtered pigs in Serbia: Serotyping, PFGE-genotyping and antimicrobial resistance.

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.