A Large-Scale Outbreak of Trichinellosis from Infected Wild Boar Meat in Croatia and the Role of Real-Time PCR Assays in Confirming the Source of the Disease.

BACKGROUND: Trichinellosis in Croatia posed a significant health concern during the 1990s, followed by a notable improvement in the epidemiological situation. However, in 2017, there was a resurgence, with 37 recorded cases in 3 outbreaks and 3 sporadic cases. The source of this epidemic was homemade meat products derived from wild boar meat, leading to 26 infections. METHODS: At the beginning of the outbreak and during the treatment of the patients, the medical and epidemiological records prepared throughout the investigation and over the course of patient treatment were reviewed. The recovery of the first-stage (L1) larvae from suspect meat products was achieved by artificial digestion. The molecular identification of the isolated larvae was performed by multiplex PCR. The molecular identification of the meat used to prepare the meat products was performed by real-time PCR assays. RESULTS: The epidemic started in early 2017. In total, 71 exposed persons were documented: 26 with clinical symptoms and 3 hospitalised in two cities in different counties. The L1 burden in three different meat products was from 5.25 to 7.08 larvae per gram (LPG), and T. spiralis was determined as the aetiological agent of the outbreak. The molecular and biological identification confirmed that implicated meat products were made solely from wild boar meat. CONCLUSIONS: Although trichinellosis is no longer a frequent occurrence in Croatia, several cases are still registered nearly every year. Wild boar meat poses an important risk factor for human health if compulsory testing is not conducted before consumption, especially if the meat products are consumed without proper thermal processing.

Mycobiota and Mycotoxin Contamination of Traditional and Industrial Dry-Fermented Sausage Kulen.

The aim of this study was to identify and compare surface mycobiota of traditional and industrial Croatian dry-fermented sausage Kulen, especially toxicogenic species, and to detect contamination with mycotoxins recognized as the most important for meat products. Identification of mould species was performed by sequence analysis of beta- tubulin and calmodulin gene, while the determination of mycotoxins aflatoxin B1 (AFB1), ochratoxin A (OTA), and cyclopiazonic acid (CPA) was carried out using the LC-MS/MS (liquid chromatography-tandem mass spectrometry) method. The results showed a significantly higher number of mould isolates and greater species (including of those mycotoxigenic) diversity in traditional Kulen samples in comparison with the industrial ones. P. commune, as a potential CPA-producer, was the most represented in traditional Kulen (19.0%), followed by P. solitum (16.6%), which was the most represented in industrial Kulen samples (23.8%). The results also showed that 69% of the traditional sausage samples were contaminated with either CPA or OTA in concentrations of up to 13.35 µg/kg and 6.95 µg/kg, respectively, while in the industrial samples only OTA was detected (in a single sample in the concentration of 0.42 µg/kg). Mycotoxin AFB1 and its producers were not detected in any of the analysed samples (

A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather.

To investigate into the T-2 and HT-2 toxin occurrence, 240 samples of unprocessed cereals (maize, wheat, barley, and oats) were sampled from different fields located in three Croatian regions during 2017-2018. In all samples, sum concentrations of T-2/HT-2 toxin were determined using the ELISA method, while the LC-MS/MS was used as a confirmatory method for both mycotoxins in positive samples (>LOD) and the establishment of T-2 over HT-2 toxin ratios. The results showed oats to be the most contaminated cereal, with T-2/HT-2 toxins detected in 70.0% of samples, followed by barley (40.9%), maize (26.8%) and wheat (19.2%), with the mean T-2/HT-2 ratio ranging from 1:2.7 in maize to 1:4.4 in oats. Sum T-2/HT-2 concentrations in two maize samples were higher than the indicative level recommended by the European Commission, necessitating subsequent investigations into the conditions under which these poorly investigated mycotoxins are produced. Statistically significantly (p < 0.05) higher concentrations of T-2/HT-2 toxin were determined in oats throughout study regions as compared to those found in wheat, but not maize and barley, while the concentrations of these mycotoxins were related to the regional weather in Croatia.

Characterization of Traditional Croatian Household-Produced Dry-Fermented Sausages.

Characterization of five types of traditional Croatian dry-fermented sausages produced by family farms was performed via identification of superficial mycobiota, physicochemical, sensory, instrumental color, fatty acids & fat quality indices. Detailed characterization of these sausages aimed to achieve standardization of their production and composition and to establish and/or improve their specification protocols. Traditional sausages varied significantly (p < 0.05) in all analyzed parameters except for the number of mold isolates. Sausages coming from eastern Croatia had a greater mold species diversity, with the highest number of isolated mycotoxigenic species in Slavonian domestic sausage. Sensory evaluation showed good acceptability of all sausages. According to health recommendations, Kulenova Seka showed the most representable values for most of fat quality indices. The results suggest the need for certain modifications in fat & fatty acid composition and, to a lesser extent, in salt content, however not at the expense of product safety, quality and acceptability.

Trichinella spp. in wild boar (Sus scrofa) populations in Croatia during an eight-year study (2010-2017).

Wild animals represent a constant source of Trichinella spp. infections for domestic animals and humans. To date, four species of Trichinella have been isolated in wild boar populations in Europe: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in addition to several mixed infection types and one hybrid formation between T. britovi and T. spiralis. Meanwhile, insufficiently thermally processed wild boar meat has been reported to be a source of trichinellosis in humans in several European countries. In Croatia, there have been no reported or proven cases of trichinellosis caused by wild boar meat consumption. The aim of this study was to obtain data on the prevalence of Trichinella species present in Croatia and to anticipated the potential risk of infection for humans in specific Croatian regions based on information obtained over an eight-year surveillance period. A veterinary inspection of wild boar carcasses for Trichinella larvae in Croatia has been mandatory since 1989, and the artificial digestion method was introduced as a compulsory test for wild boar samples in 2008. Based on the official data submitted to the Ministry of Agriculture, Directorate of Veterinary Services, in the period 2010-2017, 303 of 183,184 (0.17%) wild boar meat samples tested positive for Trichinella spp. Infected wild boar were found in 18 of 21 counties. Of these positive samples, 85 were submitted by the authorised veterinary inspectors to the National Reference Laboratory for further examination. The intensity of infection in muscle samples was 0.04-152.66 (mean: 23,37) larvae per gram, and Trichinella species were identified as T. spiralis, T. britovi, T. pseudospiralis and T. spiralis + T. britovi. Genetic analysis of T. pseudospiralis isolates demonstrated their belonging to the Palaearctic population.

Survey of T-2/HT-2 toxins in unprocessed cereals, food and feed coming from Croatia and Bosnia & Herzegovina.

The aim of this study was to investigate into the level of T-2/HT-2 toxins in different unprocessed cereals (n=201), as well as in marketed cereal-based products (n=58), feed components (n=191) and feedstuffs (n=91) coming from Croatia and Bosnia & Herzegovina. The number of positive samples of unprocessed cereals for food production (>LOD) ranged from 30.4% in barley to 68.8% in oat whereas for feed components ranged from 26.9% in wheat to 86.1% in oat. The maximal values found in unprocessed oat and oat-based feed components were 304.2µg/kg and 521.0µg/kg, respectively. As for final products, the highest T-2/HT-2 concentrations were determined in oat flakes (89.4µg/kg) and calf feed (129.3µg/kg). Despite of the increased T-2/HT-2 concentrations found in some of the samples, the obtained values were unanimously lower than the indicative levels given as recommendations above which further investigations should be necessary performed.

Moulds and mycotoxins detected in the regional speciality fermented sausage 'slavonski kulen' during a 1-year production period.

This study investigated the surface-to-interior mycotoxin contamination of the regional speciality meat product 'slavonski kulen' that occurred during a 12-month period and identified moulds present on its surface during the final production stage. In total, 15 pieces of slavonski kulen were produced and sampled at production months 0, 3, 6, 9 and 12 (three samples per piece). Concentrations of aflatoxin B1 (AFB1) and ochratoxin A (OTA) were determined using the ELISA method, while the identification of moulds made use of both traditional and molecular methods. A 12-month-long production of dry-fermented sausages under controlled conditions that did not include product cleaning at any point caused significant mycotoxin contamination, with determined mean AFB1 and OTA concentrations of 11.79 ± 2.34 and 16.13 ± 3.32 µg kg-1, respectively. Significantly higher mycotoxin levels were observed in the external layers of the products in comparison with their interior and, even more so, central parts. In total, five Penicillium species (P. polonicum, P. terrigenum, P. solitum, P. jugoslavicum and P. corylophilum) and two Aspergillus species (A. versicolor and A. sydowii) were isolated. Moulds responsible for the production of AFB1 and OTA in the later production stages had probably been overgrown by non-toxicogenic moulds, so that the former did not last until the end of the sausages' production process and were therefore not identified.

Citrinin in cereals and feedstuffs coming from Croatia and Bosnia & Herzegovina.

The aim of this study was to investigate the occurrence of citrinin in different cereals (n = 341) and feedstuffs (n = 67) coming from farms and feed factories established in Croatia and Bosnia & Herzegovina. The highest mean citrinin concentration (103 ± 102 µg/kg) was observed in cereals sampled in Bosnia & Herzegovina during 2014, although significantly different levels between the two countries were not observed. Across the cereal samples, the maximal citrinin concentration was determined in wheat (429 µg/kg), while across the feedstuffs, the highest concentration was found in pig feed (63 µg/kg). Despite of the increased citrinin levels found in some samples, especially wheat, the obtained values cannot be compared against the maximum limits, since no such levels are stipulated under the applicable legislation. But, given that data on citrinin are very scarce, they can serve as an indicator of cereal and feed contamination in this part of Europe.

Working group for trichinellosis--a way of systematic prevention, control and eradication of trichinellosis in the Republic of Croatia.

At the end of the last century, human trichinellosis was an important public health problem in the eastern parts of Croatia. Moreover, the majority of clinically infected people were registered in Vukovar-Srijem County (up to 60% of all human cases registered in Croatia). Also, 95% of all Trichinella positive swine carcasses originated from Vukovar-Srijem County. Beside the health threat, trichinellosis implied not only notable economic expenses but also threatened to endanger traditional way of life and eating habits. In order to reduce all negative consequences of the disease, a multidisciplinary Working group for trichinellosis was founded. The group consisted of scientists and experts from different fields of work, who helped and significantly contributed to minimizing the threats of trichinellosis as well as to maintaining and preserving the method of traditional processing and consumption of swine meat. The members, the methods and the results of the Working group activities will be discussed in this paper.

Bee community as a source of energy in the production of food, honey-plants in the ecosystem of Croatian Forests' hunting grounds.

In addition to the process of photosynthesis, the bee community is the main source of energy in food production, honey-plants used by game and people in the hunting grounds ecosystem. It is a well-known fact that thousands of plant species depend on the presence of bee communities (pollination, fertilization). In this paper we studied the presence of the bee community in the hunting grounds of Croatian Forests, and their influence on the number of game (wild pigs), as well as the quality of honey, honey plants in the hunting grounds used by people and game. We established the total number of game (wild boars). The honey quality parameters were determined using the Harmonised methods of the European Honey (Bogdanov et al., 1997) and the pollen analysis by were conducted according to Harmonised methods of melissopalynology (Von der Ohe et al., 2004). Research results indicate that the presence of the bee community influences the number of wild boars from 3-18%, and the quality of honey is in line with the European and world standards. The SAS/STAT package was used for the statistical analysis (SAS Institute Inc., 2000). The significance of the differences among the groups was determined by Duncan test.

Influence of keeping pheasants in captivity vs. nature on the biological value of meat and its use in human nutrition.

The life of game birds (pheasants) in nature is coupled with a number of difficulties in all seasons of the year. This refers to finding food, breeding, laying eggs, raising the young, fleeing from their natural enemies and lack of protection from unfavorable climatic conditions. The pheasants that live in captivity--aviaries for pheasants--do not have such difficulties--they are fed regularly by quality feed for pheasants, they are protected from bad weather and natural enemies. Our research was aimed at determining the biological value of meat of pheasants grown in the two different settings--in captivity and in nature. The highest weight achieved wild pheasant males (1232.4 +/- 147.36 g). The differences between tested pheasant groups were statistically very high significant (P < 0.001). The differences between groups related to breast weight and tights with drumsticks weight were statistically very high significant (P < 0.001). Between breast parts (%) and legs parts (%) were notified very high (P < 0.001) i.e. high (P = 0.002) differences. The highest weight breast muscles and tights with drumsticks had wild pheasants (282.6 +/- 63.53 g i.e. 206.2 +/- 37.88g). Wilde pheasants had lower part (%) and lighter (g) skin with subcutaneous fatty tissue on breasts. Female pheasants cultivated on both ways had higher skin part (%) and subcutaneous fatty tissue in tights with drumsticks. Related to chemical composition of breast muscles is established statistically significant differences (P < 0.001 i.s. P = 0.040)) in part of Ca (%) and P (%). In wild pheasant tights with drumsticks muscles established statistically very significant (P < 0.001) higher part of moisture, protein and Ca, i.e. statistically very high significant (P < 0.001) lower part of fat and energetic value. Research results indicate that the quality of meat of pheasants grown in nature has higher biological value than the meat of pheasants kept in aviaries, which means it has advantages in human nutrition.

Microbial and physicochemical succession in fermented sausages produced with bacteriocinogenic culture of Lactobacillus sakei and semi-purified bacteriocin mesenterocin Y.

The influence of the bacteriocinogenic culture Lactobacillus sakei (10(5)/g) and semi-purified bacteriocin mesenterocin Y (2560AU/kg) on the safety and quality of traditional Croatian fermented sausages was investigated. The addition of Lb. sakei and/or mesenterocin Y reduced microbial counts (P<0.05) in the final products. After 28 days of ripening, coagulase-negative cocci decreased 1.5-2.0log, yeasts 1.2-1.4log and enterococci 1.7-2.7log. In the case of the addition of Lb. sakei, the lactic acid bacteria count was significantly (P<0.05) higher at day 7 of ripening, and was accompanied by a lower pH and a higher amount of lactic acid (P<0.05). In the final product the amount of acetic acid was significantly lower. More intensive proteolysis and an increase in ammonia content were found at the beginning of fermentation, and in the second phase of ripening in the control samples, respectively. The free fatty acid concentration was significantly lower during the entire ripening process compared to the control (P<0.05). Semi-purified mesenterocin Y did not affect the sensory properties of the sausages, whilst the addition of Lb. sakei enhanced them.