Survey among European and Canadian feed control units on monitoring packaging material residues in feed by microscopy analyses

Macro- and microscopic evaluation of feed includes detection of animal proteins, botanical ingredients and impurities, and prohibited ingredients such as packaging material (PM), according to Regulation (EC) 767/2009. In addition, detection of micro-plastics (possible degradation products of some of the PM) is getting attention. PM can harm animals or disturb their feed intake, pollute the environment, and are considered as undesired impurities in feeds. These materials do not consist of a definite molecule, group of molecules, living species or definite bodies. They can be plastic foil, hard plastic, metal pieces, paper, wood or some combination of materials. Their features (sharp, pointed) can be as important as the material itself. This is a typical topic for microscopy detection and evaluation. This short review presents the work done on detection of PM in 15 onitoring entities (institute, laboratories). Since 2011, some institutes have analysed more than 20 samples each year and the incidence of non-compliant samples will be presented here. Thirteen out of 15 entities have an active monitoring, whereas others have passive surveillance (done while performing other microscopy analyses). The protocols used by the different entities depend on sample types and analysts, highlighting a need for harmonisation

Whole genome sequencing of Vibrio harveyi from different sites in the Mediterranean Sea providing data on virulence and antimicrobial resistance genes

Vibriosis caused by Vibrio harveyi is an important bacterial infection in the Mediterranean farming of European seabass and gilthead sea bream. Genomic information about V. harveyi contributes to understanding its pathogenesis and virulence. Sixteen V. harveyi strains obtained from diseased seabass and sea bream from Croatia, France, Italy, Spain, Tunisia, and Turkey were sequenced using ONT MinION and Illumina HiSeq. The strains were sequenced, de novo assembled, and the genomes analysed for the presence of plasmids, virulence, and antibiotic resistance genes. Antibiotic resistance genes were compared with in vitro susceptibility testing using disc diffusion and microdilution methods. All studied strains had two circular chromosomes, and twelve strains had one to five circular plasmids. A total of 171 virulence genes were determined using the Virulence Factors of Bacterial Pathogens database (VFBD), 150 virulence genes were present on average in each genome and the distribution of virulence genes was similar across strains. Most of the reported virulence genes have been previously described in bacteria of the genus Vibrio, while genes sitA (4/16), sitB (3/16), sitC (4/16), and sitD (3/16) important for iron transport, and the ast gene (10/16) that encodes a heat-stable cytotoxic enterotoxin, were unique in our studied strains. Using the Comprehensive Antibiotic Resistance database (CARD), it was noted that all strains carried the CRP gene encoding resistance to macrolides, fluoroquinolone, and penams, but showed sensitivity to enrofloxacin and florfenicol using in vitro tests. The adeF gene coding for resistance to fluoroquinolones and tetracyclines was detected in lower percentage in thirteen strains was supported with detected sensitivity to florfenicol and oxytetracycline using in vitro tests. The E.coli parE gene was detected in eleven strains that showed sensitivity to florfenicol and oxytetracycline. The gene APH(3”)Ib responsible for resistance to aminoglycosides was present in one strain, though all strains showed susceptibility to gentamicin by in vitro testing. The Tet(D) gene coding for resistance to tetracyclines was found in a single strain showing reduced sensitivity to oxytetracycline. All tested strains were resistant to ampicillin by in vitro testing in line with previously described innate resistance. Phylogenetic analysis based on whole genome sequences showed a clear separation and clustering of the tested strains from the Mediterranean region. The new genomic data and knowledge provided in this study increase the understanding of the pathogenicity and antimicrobial resistance patterns of V. harveyi strains in the Mediterranean region, enabling a common regional approach to prevention and mitigation of the disease

An overview of tick-borne encephalitis epidemiology in endemic regions of continental Croatia, 2017-2023

Tick-borne encephalitis (TBE) represents an important public health problem in Europe. We analyzed the epidemiology of TBE based on data from humans, animals, and Ixodes ricinus ticks in endemic regions of continental Croatia. In the period from 2017 to 2023, cerebrospinal fluid (CSF) and serum samples of 684 patients with neuroinvasive diseases, 2240 horse serum samples, and 300 sheep serum samples were tested for TBEV. In addition, 8751 I. ricinus ticks were collected. CSF samples were tested using RT-PCR. Serological tests (serum, CSF) were performed using commercial ELISA, with confirmation of cross-reactive samples by a virus neutralization test. Eighty-four autochthonous human TBEV cases were confirmed. The majority of patients were in the age group of 40–69 years (58.3%) with a male predominance (70.2%). TBE showed a bimodal seasonality with a large peak in April–August and a small one in October–November. In addition to humans, TBEV IgG antibodies were found in 12.2% of horses and 9.7% of sheep. Seasonal tick abundance corresponds to the reported number of human infections. Continental Croatia is still an active natural focus of TBE. Continuous monitoring of infections in humans, sentinel animals, and ticks is needed for the implementation of preventive measures

Evaluation of lipid profiles in selected fresh and dry-cured game meats – a comparative approach

This study examined and compared the fatty acid (FA) composition and fat quality indices of four types of wild game meat (deer, roe deer, mouflon, and wild boar) from Croatia, and two types of dry-cured game meat products (deer and wild boar) available on the market, in relation to their impact on consumer health. FAs were analysed by gas chromatography coupled with a flame ionization detector, and fat quality indices were calculated based on the determined FAs. Wild boar meat clearly differs from other game meat (ruminants), due to its higher fat content and higher proportion of monounsaturated fatty acids (MUFA) compared to saturated fatty acids (SFA). Palmitic and stearic acids made up the greatest proportion of the SFA component, with stearic acid most represented in mouflon and roe deer meat, and palmitic acid predominating in wild boar and deer meat. Oleic acid was the largest component of MUFAs, with wild boar meat having the significantly highest and deer meat the lowest values. Linoleic acid and α-linolenic acid made up the largest proportion of polyunsaturated fatty acids (PUFAs), with the highest proportion of α-linolenic omega-3 acid found in deer meat. The determined PUFA/SFA ratio was lower, while the thrombogenic index was above the recommended values. Wild boar meat, along with roe deer and mouflon meat, was compliant with the recommendations concerning hypo- to hypercholesterolemic fatty acids and atherogenic indices. The roe deer meat was also acceptable in terms of the n-6/n-3 ratio, along with deer meat, for which that was the only favorable index. According to health recommendations, roe deer meat showed the most favourable values for most fat quality indices, while deer meat showed the least favourable values. When comparing fresh meat and dry-cured products from the same species, no significant differences were observed

Comparison of the Nutritional Composition of Meat Products Derived from Croatian Indigenous Pig Breeds

There is a growing interest in the preservation of indigenous pig breeds, as they serve as a valuable genetic reserve. Pork meat products are widely consumed due to their desirable flavor, which is largely influenced by their chemical composition and the production processes employed. The aim of this study was to characterize and compare the nutritional composition, mineral content, and fatty acid profile of meat products derived from indigenous Croatian pig breeds. Three types of meat products, including bacon, dry-cured ham, and dry-fermented sausages, originating from the Turopolje pig, Black Slavonian pig, and Banijska šara, were collected and analyzed for proximate composition, fatty acid profile, and mineral content. Concerning the proximate analysis, statistically significant differences were found in the water and fat content in bacon and dry-fermented sausages, while the mineral analysis revealed differences in iron content. The fatty acid profile of the tested products was found to be in accordance with previously reported data. The results indicated similarities in chemical composition, mineral content, and fatty acid profile between meat products from different pig breeds; however, performing PCA analysis revealed that the major influence on product and breed characterization could be attributed to differences in fatty acid compositio

First report on the presence of potentially pathogenic Vibrio parahaemolyticus in farmed mussels (Mytilus galloprovincialis) from the Istrian aquatorium by microbiological and molecular methods

Microbiological and molecular PCR techniques were used to analyse 328 samples of mussels (Mytilus galloprovincialis) cultured in Istrian waters. Molecular testing was more effective than microbiological testing, detecting the presence of the bacterium Vibrio parahaemolyticus in 21.34% of samples, compared to 2.44% detected through microbiological methods. The probability of detecting the presence of this bacterium was 7.19 times higher when the sea surface temperature was above 15°C, while thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) positive samples were detected at sea temperatures above 22.5°C and 24.7°C, respectively. TDH- and TRH-positive samples constituted 10% of the total V. parahaemolyticus-positive samples. As there is no legal obligation to monitor the presence of this bacterium, consuming raw or insufficiently cooked mussels can lead to illness

The influence of drying on the nutritional, microbiological, and sensory value of Diospyros kaki L. (Japanese permsimmon) consumed in Herzegovina

Diospyros kaki L. (Japanese persimmon) has been introduced from Asian countries to Herzegovina, where it is mostly consumed fresh. This study investigates the differences in nutritional, microbiological, and sensory values of persimmons consumed in one of the Herzegovinian regions, following drying processes at 70 °C/12 h, 85 °C/10.5 h, and 100 °C/5.5 h, together with their effects on fruit texture, mass, and composition. The notable increase in sugar content emphasises the potential of dried Japanese persimmon as a nutritious snack. No significant increase in microorganism representation was observed across all samples. Persimmons dried using a dehydrator exhibited the most preserved nutritional properties, and were rated highest in sensorial evaluations. However, due to its high water content and browning, the sample dried at 100 °C in an autoclave was excluded from sensory testing

High genetic diversity yet weak population genetic structure in European common terns

The common tern (Sterna hirundo) is a migratory seabird experiencing a decline in breeding pairs across several European populations due to various threats, including habitat destruction and human disturbance. This study investigates the population genetic structure and diversity of common terns sampled extensively across three European areas—Northern, Southern Inland and Southern Marine—during the breeding seasons, using 18 microsatellite markers and a mitochondrial DNA control region fragment. High genetic diversity was found in both types of markers, with the Southern Marine group showing the lowest overall diversity, although signals of possible population bottlenecks were detected in all groups. Various analyses indicated that population genetic structure was weak or absent, suggesting high gene flow among groups. The low genetic differentiation is likely influenced by distinct migration patterns, particularly between Southern Inland and Marine groups. Our results suggest that geographical distance between breeding colonies had minimal effect on population genetic structure. Further studies with tracking devices are needed to clarify how migration dynamics impacts genetic structure in common terns, while conservation efforts should focus on securing multiple breeding sites and currently unoccupied areas to increase options for habitat selection, supporting the species’ genetic diversity and long-term resilience

Spatiotemporal Analysis of Stranded Loggerhead Sea Turtles on the Croatian Adriatic Coast

This study investigates the spatiotemporal trends of loggerhead turtles along the Croatian Adriatic coast by using stranding data and post-mortem analyses. Information on 620 loggerhead turtles (Caretta caretta), collected in the period between 2010 and 2022, has been analysed. Seasonal stranding variations reveal distinct patterns, indicating season-specific abundance and age-specific mortality in different areas, particularly in the key neritic habitat of the northern Adriatic. The analysis identifies four critical areas in the northeast and central Adriatic showing high stranding densities and provides regional managers with a tool with which to effectively conserve and manage this species. Fishing-induced mortality, collision with vessels, and potential cold stunning are identified as major threats to loggerhead turtles. Post-mortem investigations reveal that longline fishing gear and collisions with vessels are significant age-specific mortality contributors, underscoring the need for targeted conservation efforts in high-risk areas. The study acknowledges potential biases in strandings records but highlights the importance of post-mortem investigations in understanding mortality causes. The findings provide valuable insights for improving conservation strategies, emphasizing the importance of focused surveillance and conservation efforts in identified high-risk locations to mitigate human-turtle interactions

Inter-laboratory comparison of eleven quantitative or digital PCR assays for detection of proviral bovine leukemia virus in blood samples

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts