Bartosz Sell

Aujeszky’s disease (AD), caused by suid herpesvirus 1 (pseudorabies virus, PRV), is a highly contagious infection primarily affecting swine, with wild boars serving as an important reservoir in Europe. Spillover infections in non-suid species, including carnivores, are rare but typically fatal and of epidemiological concern. This study presents the first case of AD in a grey wolf (Canis lupus) in Central Europe with genomic characterization. The 8-month-old wolf was found in the Carpathians (SE Poland), moribund with acute neurological signs, and euthanized for animal welfare reasons. Necropsy revealed no pathognomonic gross lesions. Molecular analyses of tissues confirmed the presence of PRV DNA using real-time PCR, and virus isolation was successful. Genomic analysis revealed that the PRV isolate clustered within genotype I, the predominant circulating genotype in Europe. However, due to the limited availability of reference PRV genome sequences from European wildlife, the precise geographic origin and transmission pathways of this strain could not be fully resolved. In the presented case, wild boars were considered a possible source of infection. This highlights the potential for PRV transmission to apex predators. This study emphasizes the importance of systematic surveillance of PRV in wildlife and the need for expanded genomic databases of PRV strains. Full-genome sequencing is crucial for improving the understanding of PRV transmission.

Introduction: Sedatives have been used for a long time as animal tranquillisers to prevent stress and weight loss during their transportation. The proper determination of these substances in food of animal origin is essential for consumer safety. Material and Methods: A 1 g portion of pig or cow urine or homogenised kidney was mixed with acetonitrile, sodium chloride was added, and the solution was further mixed and then centrifuged. The supernatant was transferred to a new centrifuge tube with primary and secondary amine, octadecylsilane and ZrO₂, and mixed rapidly. The filtered solution was evaporated under a nitrogen stream. The residue was dissolved in 200 μL of acetonitrile, centrifuged with filters and then transferred to vials. Samples were analysed by high-performance liquid chromatography–tandem mass spectrometry. Results: The decision limit for confirmation was calculated at 2.5 μg kg⁻¹ for all sedatives with relative standard deviation repeatability and reproducibility below 20%. Conclusion: The validation results showed that this method meets the pertinent EU criteria for such methods and is suitable for sedative analysis in urine and kidney matrices.

Bovine foamy virus (BFVbta) displays a very high degree of cell-associated replication which is unprecedented even among the other known foamy viruses. Interestingly, recent studies have shown that it can in fact adapt in vitro to high-titer (HT) cell-free transmission due to genetic changes acquired during repeated rounds of cell-free BFVbta passages in immortalized bovine MDBK cells. Molecular clones obtained from the HT BFVbta Riems cell-free variant (HT BFVbta Riems) have been thoroughly characterized in MDBK cell cultures However, during recent years, it has become increasingly clear that the source of the host cells used for virus growth and functional studies of virus replication and virus–cell interactions plays a paramount role. Established cell lines, mostly derived from tumors, but occasionally experimentally immortalized and transformed, frequently display aberrant features relating, for example. to growth, metabolism, and genetics. Even state-of-the-art organoid cultures of primary cells cannot replicate the conditions in an authentic host, especially those concerning cell diversity and the role of innate and adaptive immunity. Therefore, to determine the overall replication characteristics of the cloned wt and HT BFVbta Riems variant, we conducted a small-scale animal pilot study. The replication of the original wt BFVbta Riems isolate, as well as that of its HT variant, were analyzed. Both BFVbta variants established infection in calves, with proviruses in peripheral blood mononuclear cells and induced Gag-specific antibodies. In addition, a related pattern in the host innate immune reaction was detected in the peripheral blood leukocytes of the BFV-infected calves. Surprisingly, an analysis of the Gag sequence two weeks post-inoculation revealed that the HT BFVbta variant showed a very high level of genetic reversion to the wild type (parental BFVbta genotype).

The white-tailed eagle (Haliaeetus albicilla) is strictly protected in Poland due to its threat of extinction. This study’s main goal was to assess their exposure to indirect poisoning by anticoagulant rodenticides (AR). This study presents the investigation results of 40 white-tailed eagles’ suspected poisoning cases in the years 2018–2020 in Poland. In all tested liver samples, using a liquid chromatography–mass spectrometry method, at least one of the AR (bromadiolone, brodifacoum, difenacoum, flocoumafen) was detected and confirmed. The other tested AR compounds (chlorophacinone, coumachlor, coumatetralyl, difethialone, diphacinone, warfarin) were not detected. The mean concentration of the sum of rodenticides was 174.4 µg/kg (from 2.5 to 1225.0 µg/kg). In 20 cases, the sum concentration was above 100 µg/kg and in 10 cases it was above 200 µg/kg. Interpretation of cases of AR poisonings should take into account their concentration in the liver, anatomopathological lesions, circumstances of death/finding of the animal, and elimination of other possible causes of poisoning. Based on this study, AR was the direct cause of death in 10% of incidents. Extensive use of rodenticides generates a high risk of poisonings of white-tailed eagles in Poland.

The purpose of this study was to assess the formation of chloramphenicol metabolites in primary turkey and rat hepatocyte cultures and human hepatoma (HepG2) cells and nonhepatic, Balb/c 3T3 fibroblasts. Additionally, the cytotoxicity of the drug was assessed through three biochemical endpoints: mitochondrial and lysosomal activity and cellular membrane integrity after 24 and 48 h exposure. The two metabolites of the drug, chloramphenicol glucuronide and nitroso-chloramphenicol, were detected to the greatest extent in both primary hepatocyte cultures by liquid chromatography–tandem mass spectrometry. Toxic nitroso-chloramphenicol was the main metabolite in the primary turkey hepatocyte cultures, but it was not in the primary rat hepatocyte cultures. The most affected endpoint in turkey and rat hepatocyte cultures was the disintegration of the cellular membrane, but in the cell lines, mitochondrial and lysosomal activities underwent the greatest change. The primary hepatocyte cultures represent valuable tools with which to study the species differences in the biotransformation and toxicity of drugs. To the best of our knowledge, this is the first report of differences in chloramphenicol metabolism in primary turkey and rat hepatocyte cultures.

Strychnine is an alkaloid with strong toxic properties. Poisoning results in muscular contractions and death through asphyxiation. Intentional or accidental poisonings with strychnine occur mainly in small animals, especially dogs and occasionally cats. Strychnine can be detected in the liver or stomach contents. Unfortunately, the determination of strychnine in these matrices, especially in postmortem examination, is subject to a significant matrix effect that makes it difficult to confirm the presence of the substance being determined. Therefore, we developed a new liquid chromatography method combined with mass spectrometry. One-gram homogenized samples were extracted and partitioned after adding acetonitrile and 5-mol solution of ammonium acetate. After extraction, the samples were analyzed using high-pressure liquid chromatography-MS/MS/MS. The results of validation fulfil the requirement of the confirmatory criteria according to SANTE/11945/2015 regarding apparent recoveries (98.97% to 104.0%), repeatability (2.9%–4.1%), and within-laboratory reproducibility (3.3%–4.6%). The method can be successfully applied to confirm strychnine poisoning cases.

A simple method for the determination of chloramphenicol in 22 matrices was prepared based on the QuEChERS and HPLC-MS/MS combination. Following a hydrolysis step, the homogenized samples were extracted and partitioned after adding sodium chloride with acetonitrile. Chloramphenicol was analysed by HPLC-MS/MS in negative electrospray mode by monitoring the daughter ions m/z: 321→194 and 321→152. The limit of decision (CCα) was calculated at the range of 0.10 μg kg⁻¹ to 0.15 μg kg⁻¹ and detection capability (CCβ) from 0.12 μg kg⁻¹ to 0.18 μg kg⁻¹. Validation results showed that this method is suitable for the determination and confirmation of chloramphenicol in various matrices.

Reported here is a new analytical multiclass method based on QuEChERS technique, which has proven to be effective in diagnosing fatal poisoning cases in animals. This method has been developed for the determination of analytes in liver samples comprising rodenticides, carbamate and organophosphorus pesticides, coccidiostats and mycotoxins. The procedure entails addition of acetonitrile and sodium acetate to 2 g of homogenized liver sample. The mixture was shaken intensively and centrifuged for phase separation, which was followed by an organic phase transfer into a tube containing sorbents (PSA and C18) and magnesium sulfate, then it was centrifuged, the supernatant was filtered and analyzed by liquid chromatography tandem mass spectrometry. A validation of the procedure was performed. Repeatability variation coefficients <15% have been achieved for most of the analyzed substances. Analytical conditions allowed for a successful separation of variety of poisons with the typical screening detection limit at ≤10 μg/kg levels. The method was used to investigate more than 100 animals poisoning incidents and proved that is useful to be used in animal forensic toxicology cases.