Trichinella proficiency testing in Southeastern European countries.

In some Southeastern European (SEE) countries, like Serbia, Romania, and Bulgaria, trichinellosis is one of the most important foodborne zoonotic diseases. In those countries, EU regulation and local authorities require the staff of laboratories performing official controls on meat to be properly trained and to check their competence by participating regularly in proficiency testing (PT). PTs are organized by National Reference Laboratories for Trichinella of each country and involve all official laboratories testing meat. In Romania and Bulgaria, the organization of PT for the detection of Trichinella larvae in meat by Magnetic Stirrer Method (MSM) started in 2012. In Croatia and Serbia PT was first organized in 2015 and 2017, respectively. This study presents data on the performance obtained by official laboratories of SEE countries that organize PT at national level and compares the performance obtained by laboratories belonging to different countries. Results suggest that the constant participation in PT leads to an increase in the performance of participating laboratories by positively affecting the staff accuracy in sample testing by MSM. Since the percentage of recovered larvae was in some cases suboptimal (<80%) and occasionally very poor (<40%), there is room for improvement. The regular participation in PT by laboratories involved in official controls on meat intended for human consumption is fundamental to guarantee consumer safety.

Evaluation of the physicochemical, biochemical and microbiological characteristics of three Serbian traditional dry-fermented sausages.

Three Serbian traditional dry-fermented sausages, Sremski kulen (S), Lemeski kulen (L) and Petrovacka kobasica (P), were compared for physicochemical, biochemical and microbiological properties, in order to provide complex overview of their quality and safety. The sausages were produced and analyzed within two production seasons in traditional practice. Water activity, color, texture and proximate composition varied significantly (P < 0.05), particularly due to moisture, protein and fat content, what indicated differences in basic formulation of raw sausage mixture, type of casing and some technological procedures. Based on the fatty acid profile the most favorable PUFA/SFA ratio was obtained in sausage P (0.68), while the obtained results for acid value (15.2-22.4 mg KOH/g lipid) and TBARS value (0.27-0.55 mg malondialdyde/kg) indicated low level of lipid degradation and oxidation in all samples. Regarding the microbial and biogenic amines (BA) analysis, positive outcome of this research is the total absence of all pathogenic microorganisms examined, while the registered levels of BA showed a great variability among the sausages. In particular, total BA content in L attained level that is approx. 50% higher than the recommended one. Hence, special care should be implemented regarding the future production and consumption of this type of sausage. Overall results of this study may be useful to define complete distinctive features of these sausages, to determine relations among them, as well as to improve some phases of traditional manufacturing process.

Determination of toxic elements in meat products from Serbia packaged in tinplate cans.

This work aimed to examine the influence of the storage period on the content of toxic elements (As, Cd, Hg, and Pb) in five types of canned meat products regularly used in the Serbian Armed Forces. Cans of beef goulash (BG), pork ragout (PR), spam (SP), liver pate (LP), and meatballs in tomato sauce (MB), produced according to military standards and stored under regular conditions, were analyzed. Meat products were packed in tin cans made according to special requirements in terms of tin and varnish application and stored for up to 6 years. The content of toxic elements varied depending on the analyzed product. The highest average content of arsenic was in BG (10.00 µg/kg), cadmium in LP (35.91 µg/kg), and mercury and lead in PR (15.04 and 8.00 µg/kg, respectively). The average concentrations of As, Cd, Hg, and Pb in all types of canned meat products were significantly lower than the maximum permitted levels in food currently in force by local and EU legislation. The storage period did not significantly affect the level of toxic elements, although higher concentrations were found in samples stored for more than 2 years. Examination of raw materials, spices, and additives showed that the highest Cd and Pb concentrations, which can affect the total level of these elements in meat products, were found in red ground pepper (Cd above 150 µg/kg) and dish supplement (Pb of 250 µg/kg). The assessment of the weekly intake of toxic elements through canned meat showed that it is significantly lower than the values that affect adversely to human health, as determined by the FAO/WHO and EFSA. However, as there is a constant possibility of contamination of raw materials and food additives, primarily due to environmental pollution, it is recommended to monitor the content of heavy metals in food permanently and assess their risk to human health.

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.

The effects of season on health, welfare, and carcass and meat quality of slaughter pigs.

This study assessed the effects of season on health, behaviour, physiological stress parameters, and carcass and meat quality in a total of 480 slaughter pigs. The following health indicators were recorded: pneumonia, pleurisy, milk spots, and pericarditis. Behaviour was monitored during unloading (slipping, falling, turning back, reluctance to move, panting, shivering) and lairaging (panting, shivering, huddling). Blood lactate and glucose concentrations were determined at exsanguination. Performance indices (live weight, daily weight gain), carcass (carcass weight, backfat and loin thickness, lean meat content, carcass lesion score), and meat quality (pH, temperature, drip, thawing and cooking losses, colour, marbling) traits were measured postmortem. Pigs slaughtered in winter had the highest live weight, carcass weight, loin thickness, and carcass lesion score, while the lowest live weight, carcass weight, and backfat thickness were recorded in pigs slaughtered in summer. The highest lactate and glucose concentrations were recorded in pigs slaughtered in summer. The highest prevalence of red, soft, and exudative meat was recorded in pigs slaughtered in winter. Pigs slaughtered in summer had the lowest pH, the highest thawing loss, L* value, b* value, and occurrence of pale, soft, and exudative meat. Pigs slaughtered in autumn had the lowest drip loss, cooking loss, L* value, b* value, and the greatest percentage of red, firm, and nonexudative meat. In conclusion, the summer and winter temperatures compromised health and welfare and reduced carcass and meat quality in slaughter pigs, indicating that protection against heat and cold stress is not yet effective.

Identifying Physiological Stress Biomarkers for Prediction of Pork Quality Variation.

This study assessed the potential use of various physiological stress biomarkers as indicators of carcass and meat quality traits in 240 pigs subjected to the standard marketing conditions and minimal stressful antemortem handling using Pearson correlations. The most important pork quality traits (pH and temperature, water holding capacity, and color) had limited correlations with stress metabolites (lactate, glucose), stress hormones (cortisol, adrenocorticotropic hormone), stress enzymes (creatine kinase, aspartate amino transferase, alanine amino transferase), electrolytes (sodium, chloride), and acute-phase proteins (haptoglobin, C-reactive protein, albumin), indicating poor reliability in predicting pork quality. Albumin level was moderately positively correlated with live weight, hot carcass weight, cold carcass weight, and back fat thickness. Alanine amino transferase level was moderately positively correlated with live weight, hot carcass weight, and cold carcass weight. Cortisol level was moderately positively correlated with live weight, hot carcass weight, cold carcass weight, and back fat thickness, and moderately negatively correlated with the lean carcass content. Increased lactate dehydrogenase level was moderately correlated with decreased drip and cooking loss. In conclusion, lactate dehydrogenase could help pork producers predict pork quality variation, while cortisol, alanine amino transferase, and albumin could be useful in prediction of carcass quality.

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.

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.

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.

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.