Biogenic amine content during the production and ripening of Sremski kulen, Serbian traditional dry fermented sausage.

Sremski kulen is a wide diameter dry fermented sausage, produced from pork, seasoned with red spicy paprika, stuffed into pork cecum, and preserved by smoking, fermentation and drying. Due to specific ripening process, Sremski kulen is suitable for the accumulation of biogenic amines. Therefore, the aminogenesis was studied in traditionally produced Sremski kulen, taking into account the physicochemical parameters and microbial counts. The content of six biogenic amines (tryptamine, phenylethylamine, tyramine, histamine, putrescine, and cadaverine) was analyzed using high-performance liquid chromatography. The ripening process of Sremski kulen was slow followed by changes in aw and pH value as well as expressed proteolysis. The autochthonous microbiota showed pronounced decarboxylase activity. Tryptamine and phenylethylamine were detected at each examined ripening stage while histamine was not detected until the end of ripening (16.55 ± 2.33 mg/kg). Tyramine, cadaverine, and putrescine content significantly increased during the ripening period (p < .05). In the final product, cadaverine was the dominant biogenic amine (132.40 ± 5.05 mg/kg), followed by tyramine (115.80 ± 15.46 mg/kg) and putrescine (68.55 ± 2.39 mg/kg). Although the long ripening period greatly contributed to the accumulation of biogenic amines in final product, their content are not of concern from product safety aspects, but requires improvement in hygiene of production process.

Fatty acid profile as a discriminatory tool for the origin of lamb muscle and adipose tissue from different pastoral grazing areas in North Macedonia - A short communication.

The fatty acid (FA) profiles of lamb muscle and adipose tissue originating from ten different grazing areas in North Macedonia and an assessment of whether the meat origin could be distinguished on the basis of FA profile are presented. Muscle and adipose tissues of three-month-old male lambs (crossbreds of autochthonous Ovcepolian and Württemberg) reared on spring pastures were studied. Statistically significant differences in the individual FA contents, FA groups and FA ratios were observed between lamb tissues from the different geographic regions. Canonical discriminant analysis showed there was a significant linear divergence between tissues from almost all examined regions. The greatest weight in the differentiation of the different areas showed fatty acid ratios, C18:3n3, C18:1n9c and C20:5n3 for the muscle tissue, as well as C18:1n9c, C18:2n6c, C16:1 and C17:1 for the adipose tissue. This showed that the FA composition of muscle and adipose tissue obtained from lambs reared on pasture could serve as a useful indicator to aid differentiation of its geographic origin but it should be confirmed through further replicated studies.

Slaughterline records of various postmortem pathological lesions and their influence on carcass and meat quality in slaughtered pigs.

The aim of this study was to identify the prevalence of pathological lesions in pigs from small-scale farms and to determine associations between pathological lesions and hematological parameters, and carcass and meat quality in slaughtered pigs. The study was conducted on 625 pigs (~115 kg) originating from 20 small-scale farms. Any signs of pneumonia, pleurisy, pericarditis, and liver milk spots were recorded as present or absent. Complete blood count was investigated. The following carcass quality parameters were measured: live, hot and cold carcass weights, cooling loss, dressing percentage, backfat thickness, and meatiness. Meat pH and temperature were measured 45 min postmortem. Of the 625 examined pigs, 41.8% had pneumonia, 23.5% pleurisy, 2.7% pericarditis, and 29.9% liver milk spots. The presence of pathological lesions in slaughtered pigs adversely affected hematological parameters, reduced live, hot and cold carcass weights, and meatiness and had deleterious effects on meat quality (higher pH45min and higher prevalence of dark, firm and dry meat). In conclusion, this study showed a high prevalence of pathological lesions in slaughtered pigs, indicating serious health problems in smallholder pig production systems. The presence of single and, especially, multiple pathological lesions in slaughtered pigs negatively affected hematological parameters, and carcass and meat quality.

Occurrence and dietary exposure of organochlorine pesticides in common carp obtained from integrated production systems.

The aim of this study was to determine the level of the organochlorine pesticides (OCPs) in meat, liver and skin of common carp. Fish were collected in spring and in autumn from fishpond that receives purified water from slaughterhouse. OCPs, including α-HCH, ß-HCH, δ-HCH, lindane, heptachlor, aldrin, heptachlor epoxide, trans-chlordane, cis-chlordane, endosulfan I, DDE, dieldrin, endrin, DDD, endosulfan II, dichlorodiphenyltrichloroethane (DDT), endosulfan sulphate, methoxychlor, and endrin ketone, were determined by using a gas chromatography-mass spectrometry method. OCPs were present at low concentrations and only aldrin was detected in all examined tissues. The estimated daily intake for OCPs via common carp from integrated system of production was at the lower level compared with the acceptable daily intake recommended by different organisations, showing no significant risk on consumers.

Screening and Molecular Characterization of Hepatitis E Virus in Slaughter Pigs in Serbia.

Hepatitis E virus (HEV) is a zoonotic virus that can cause acute hepatitis in humans. Besides the fecal-oral route, transmission can occur by consumption of undercooked pig liver. Genotype 3 is the most frequent genotype found in Europe. Studies on HEV in slaughter-age pigs have not been conducted in Serbia so far. Pork meat production and consumption in Serbia is on average, higher than in the rest of Europe. With the aim to identify the circulating HEV genotypes, pig livers and swab samples from three pig slaughterhouses located in three different sub-regions of Serbia were collected. A nested RT-PCR was used to amplify the hypervariable HEV ORF-1 region (334 bp). The amplicons yielded in this study were sequenced, and a molecular phylogeny analysis based on the maximum likelihood method, including HEV sequences reported in several other countries, was performed. The average prevalence of HEV genotype 3 in 3-month-old pigs was 34%. Phylogenetic analysis revealed the majority of HEV amplification fragments from Serbia were grouped in four clades within sub-genotype 3a and were also genetically related to German, Italian, Slovenian, and American HEV sequences. Sub-genotypes 3b and 3j were also found in a single pig each. This study provides the first analysis of the genetic diversity and circulation dynamics of HEV in pigs at slaughterhouses in Serbia.

Trichinella spp. in wild mesocarnivores in an endemic setting.

Human trichinellosis and Trichinella infection in pigs are both still endemic in the Balkans, including Serbia. Because of the flow between the sylvatic and the domestic cycle of Trichinella spp., monitoring wildlife has been recommended for the risk assessment of Trichinella spp. infection in swine. We have previously shown the presence of Trichinella infection in wild carnivores including the wolf and the golden jackal, and here we report on Trichinella infection in several other mesocarnivore species. From a total of 469 animals collected between 1994 and 2013, Trichinella larvae were detected in 29 (6.2%, 95% CI = 4.0-8.4) animals, including 14 red foxes (4.7%), 7 wild cats (35%), 5 beech martens (4.8%), 2 pine martens (16.7%), and 1 European badger (6.25%). No Trichinella larvae were detected in the examined specimens of European polecats, steppe polecats and European otters. Species identification of the Trichinella larvae performed for 18 positive samples revealed T. spiralis in 77.8% and T. britovi in 22.2% of the isolates. Both species were detected in red foxes and wild cats. The predominance of T. spiralis in wildlife in Serbia indicates the (past or present) spillover of this pathogen from domestic to wild animals.

Molecular characterization of Listeria monocytogenes isolates from a small-scale meat processor in Montenegro, 2011-2014.

The presence of Listeria monocytogenes was evaluated in a small-scale meat processing facility in Montenegro during 2011-2014. L. monocytogenes isolates from traditional meat products and environmental swabs were subjected to a) molecular characterization b) serotyping by both multiplex PCR and next generation sequencing (NGS) c) potential antimicrobial resistance (AMR) was assessed by extraction of specific genes from NGS data and d) screening for the presence of some disinfectant resistance markers. Overall, traditional meat products were contaminated, most likely from incoming raw materials, with 4 major specific STs of L. monocytogenes (ST515, ST8, ST21, ST121) representing 4 clonal complexes (CC1, CC8, CC21, CC121) identified during the four-year period. These strains belonged to serogroup IIa which predominated, followed by IVb (ST515, CC1). The strains from environmental swabs belonged, exclusively, to ST21 and were isolated from cutting board and floor swabs in 2011. Furthermore, we found Tn6188, a novel transposon conferring tolerance to BC, to be specific to sequence type ST121. In addition, antimicrobial resistance genes mprF and fosX were present in clonal complexes CC21 and CC121, while complexes CC8 and CC1 exclusively harbored the mprF antimicrobial resistance gene.

Reduction of polycyclic aromatic hydrocarbons in common carp meat smoked in traditional conditions.

Common carp meat was smoked in traditional conditions without filter and with zeolite, granular activated carbon, and gravel filters. The aim of this study was to determine the influence of using different filters in traditional smoking conditions on 16 polycyclic aromatic hydrocarbons from the Environmental Protection Agency list (16 US-EPA PAHs). Determination and quantification of PAHs were performed by gas chromatography-mass spectrometry (GC-MS). According to the obtained results, all examined samples of common carp meat smoked in traditional conditions were safe for consumers regarding to the European Commission Regulation on PAH content. After statistical analysis, the zeolite filter appeared to be the best one based on food safety of traditional production of smoked common carp meat. Application of any of these filters to produce smoked common carp meat in traditional conditions resulted in a safer product, as lower levels of PAHs were obtained.

Influence of Environmental Conditions on Norovirus Presence in Mussels Harvested in Montenegro.

This study comprises the first systematic survey of the occurrence of Norovirus in Mediterranean mussels from harvesting areas in Montenegro coast of Adriatic Sea. Mussels may accumulate contaminants of public health concern, including pathogenic bacteria and viruses. Microbiological monitoring of harvesting areas is based on count of Escherichia coli in bivalve molluscs in the European Union. It is assumed that E. coli does not reflect contamination with enteric viruses. A structured field study was undertaken at six locations in Bay of Kotor, Montenegro, in order to investigate plausible influence of environmental factors on the variability of E. coli and norovirus (NoV). From July 2015 to July 2016, a total of 72 samples of mussels were collected in coastal harvesting areas of the Montenegro. The samples were screened for NoV of genogroups GI and GII using reverse transcription-qPCR (RT-qPCR). There were 43% NoV positive samples with higher presence of genogroup GII (74.2%). With regard to influence of environmental conditions on Norovirus presence, we have proved seasonal pattern of virus occurrence i.e., the largest number of positive samples was noticed during winter, while other physico-chemical factors were not of great significance. It was found that count of E. coli did not correlate with Norovirus prevalence. From the aspect of food safety, an upgrade of monitoring plans could lead to obtaining safer products.

Determination of heavy metals in muscle tissue of six fish species with different feeding habits from the Danube River, Belgrade-public health and environmental risk assessment.

The aim of this study was to investigate the presence of heavy metals/metalloids (Pb, Cd, Hg, Cu, Fe, Zn, As) in the muscle tissue of fish from the Danube River (two locations: Zemun and Grocka). For the purpose of heavy metal determination in fish muscle, 120 samples of six different fish species, Prussian carp, barbel, bream, carp, pike perch, and catfish were collected. For determining heavy metals, we used microwave oven digestion and atomic absorption spectrometer methods. The highest average content of Pb (0.084 ± 0.004 mg kg-1), Cd (0.082 ± 0.003 mg kg-1), Hg (0.466 ± 0.006 mg kg-1), and As (0.333 ± 0.007 mg kg-1) was found in the muscle of carp (an omnivorous fish) from Grocka, while the highest average level of Fe (13.60 ± 0.03 mg kg-1) was deposited in bream (also omnivorous) from Zemun. Also, the average Cu level (1.62 ± 0.13 mg kg-1) was the highest in catfish muscle (a carnivorous fish) from Grocka, while the highest Zn content (11.16 ± 0.17 mg kg-1) was determined in muscle of Prussian carp (an omnivorous fish) from Zemun. The highest content of heavy metals (Cu, Fe, and Zn, respectively) in muscle of the six different types of fish from both locations was symmetrically arranged by species (catfish, barbel, and Prussian carp, respectively). Concentrations of Pb, Hg, and As in the Danube River fish muscle were under the maximum residual levels prescribed by the European Union (EU) and the maximum allowed concentrations (MAC) for Serbia. On the other hand, in all fish muscle from both locations (Zemun and Grocka), higher concentrations of Cd than prescribed (MAC) were found, with the exception of bream and pike perch.

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.

A large-scale study of the Trichinella genus in the golden jackal (Canis aureus) population in Serbia.

Over the last decades the golden jackal (Canis aureus) has significantly expanded its range throughout Southeast and Central Europe, and the Balkan Peninsula is considered to be a core area of the species distribution in this part of the range. Due to its increasing number, ability of long distance movement through a wide range of landscapes and opportunistic feeding habits, the golden jackal may represent an important reservoir and transmitter of a variety of zoonotic agents, including parasites. The Balkans, Serbia included, remain an endemic area for various zoonotic parasites including Trichinella spp. Trichinella has recently been recorded in jackals in Serbia, which prompted us to carry out a large-scale survey of its prevalence, distribution and species identification in this host. In cooperation with local hunters, carcasses of a total of 738 legally hunted golden jackals were collected at 24 localities over an 11-year period (2003-2013). Analysis of tongue base tissue revealed Trichinella larvae in 122, indicating a prevalence of infection of 16.5%. No difference in the prevalence of infection was found between genders [16.2% in males and 16.9% in females (χ(2)=0.05, p=0.821)], or among the study years (G=7.22, p=0.705). Trichinella larvae were found in 13 out of the 24 examined localities. Molecular identification was performed for 90 isolates, and 64 (71.1%) larvae were identified as Trichinella spiralis and 25 (27.9%) as Trichinella britovi. Mixed infection (T. spiralis and T. britovi) was recorded in a single case. Although T. spiralis was more prevalent, T. britovi had a wider distribution, and was the only recorded species in jackal populations from the mountainous region of eastern Serbia. On the other hand, T. spiralis was dominant in jackals in the lowlands of central and northern Serbia, where domestic pigs are mostly reared. These results show that the golden jackal is involved in both the domestic and sylvatic cycle, and that it has emerged as a major host species in the sylvatic cycle of the Trichinella genus. Therefore, continued monitoring of Trichinella infection in golden jackals in Serbia and the whole of the Balkans is recommended in order to control transmission of this parasite to humans and domestic animals.

The wolf (Canis lupus) as an indicator species for the sylvatic Trichinella cycle in the Central Balkans.

Wildlife is the most important reservoir of Trichinella spp. worldwide. Although the Balkans are a recognized European endemic region for Trichinella infections, data on wildlife are scarce. To monitor the circulation of these zoonotic parasites in the Central Balkan region, the wolf (Canis lupus) was selected because of its abundance (>2,000 individuals) and because it is at the top of the food chain. A total of 116 carcasses of wolves were collected in Serbia and in the neighboring areas of Macedonia and Bosnia and Herzegovina (B&H) between 2006 and 2013. Trichinella spp. larvae were found in 54 (46.5%) wolves. The great majority (90.5%) originated from Serbia, where 52 of the 105 examined animals were Trichinella positive (49.5%; 95% confidence interval  =39.9-59.1). One positive animal each was found in B&H and Macedonia. All larvae were identified as Trichinella britovi. The high prevalence of Trichinella infection in wolves suggests that this carnivore can be a good indicator species for the risk assessment of the sylvatic Trichinella cycle in the Central Balkans.

Cadmium and mercury accumulation in European hare (Lepus europaeus): age-dependent relationships in renal and hepatic tissue.

A total of 63 European hares have been collected from five Serbian agricultural regions. The hares assayed were divided into four age groups (3-6 months, 12 months, 12-24 months, and 24-36 months) and investigated upon presence at cadmium (Cd) and mercury (Hg) in the kidney and liver. The positive significant correlation (Ps-Pearson's coefficient) between Cd concentrations in the kidney and liver within age group the 3-6 months was found (Ps = 0.81, p < 0.001). Differences between Cd content in the kidney in comparison to liver were significant within all presented age groups (p < 0.001). Differences between recorded Hg levels in the kidney were not significant between presented age groups (p > 0.05). Statistically significant differences were registered between Hg content in the liver of the hares aged 24-36 and 12 months (p < 0.05). There were no statistically significant correlations registered between Hg concentrations in the kidney and liver within any particular age group (p > 0.05). The strong statistically significant associations were registered between Cd and Hg content in the liver (Cd L/Hg L) in the age group 3-6 and 12-24 months (Cd L/Hg L, Ps = 0.94; p < 0.001 and Ps = 0.91; p < 0.001, respectively). The polynomial regression model used for graphing the observed data seems to be a method for modeling the relationship between measured Cd and Hg concentrations in the liver and kidney as first approximation for bioaccumulation in hares.

Environmental Cd and Zn concentrations in liver and kidney of European hare from different Serbian regions: age and tissue differences.

A total of 84 European hares collected from eleven Serbian regions investigated upon cadmium (Cd) and zinc (Zn) presence. Strong statistically significant correlations between Cd concentrations in kidney and liver were registered in animals older than 12 months. Significant differences between Zn concentrations in liver in comparison to kidney were found within every single age group with exception of the oldest. Negative correlation (Ps-Pearson's correlation) between Zn and Cd concentrations were found in liver samples within the age group of 12 months (Ps = -0.67, p = 0.004).