Carvacrol is an essential oil derived from oregano that is used as a natural additive to improve the efficiency of livestock nutrition. Residues of natural additives such as carvacrol should be monitored in food of animal origin to ensure consumer safety. The aim of this study was to appraise the quick, easy, cheap, effective, rugged and safe (QuEChERS) approach coupled with liquid chromatography and mass spectrometry as a means of carvacrol analysis in chicken tissue. A 5 ± 0.05 g portion of plasma, lung, muscle and liver was mixed for 15 min with 5 mL of 1-butanol and 20 mL of water, then centrifuged. A 0.5 mL volume from the top layer was transferred, then 60 mg of octadecylsilane sorbent, 30 mg of primary and secondary amine and 200 mg of MgSO₄ were added. The extract was mixed and centrifuged. The top layer was filtered and then transferred to an autosampler vial for analysis by high-performance liquid chromatography–tandem mass spectrometry. The limit of detection was calculated at 0.06 μg g⁻¹ and the limit of quantification was 0.2 μg g⁻¹, with relative standard deviation repeatability and reproducibility below <20%. The validation results showed that this method could be a good alternative to determination of carvacrol by gas chromatography and is suitable for carvacrol analysis in different matrices.

Carvacrol is an essential oil derived from oregano that is used as a natural additive to improve the efficiency of livestock nutrition. Residues of natural additives such as carvacrol should be monitored in food of animal origin to ensure consumer safety. The aim of this study was to appraise the quick, easy, cheap, effective, rugged and safe (QuEChERS) approach coupled with liquid chromatography and mass spectrometry as a means of carvacrol analysis in chicken tissue.

Clinical mastitis (CM) is one of the most common diseases of dairy cows globally, has a complex aetiology and recurs easily. Staphylococcus aureus is a frequently isolated pathogen responsible for bovine mastitis and remains difficult to eradicate. To characterise the transcriptional profiles of dairy cows infected by S. aureus, we performed an RNA-seq analysis of peripheral blood leukocytes in lactating Chinese Holstein dairy cows with CM and did the same with healthy cows’ samples as controls. A total of 4,286 genes were detected in the CM cases infected with S. aureus which were differentially expressed compared to the controls, 3,085 of which were upregulated, the remainder being downregulated. Notably, we observed that some differentially expressed genes (DEGs) had strong protein–protein interaction. Of these, six downregulated DEGs (AKR1C4, PTGS2, HNMT, EPHX2, CMBL, and IDH1) were involved in the metabolic pathway, while eight upregulated DEGs (VWF, GP9, MYLK, GP6, F2RL3, ITGB3, GP5, and PRKG1) were associated with the platelet activation pathway. The transcriptome dataset of CM cases would be a valuable resource for clinical guidance on anti-inflammatory medication and for deeper understanding of the biological processes of CM response to S. aureus infection, and it would enable us to identify specific genes for diagnostic markers and possibly for targeted therapy.

Clinical mastitis (CM) is one of the most common diseases of dairy cows globally, has a complex aetiology and recurs easily. Staphylococcus aureus is a frequently isolated pathogen responsible for bovine mastitis and remains difficult to eradicate.

Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan. The patient was a female toy poodle presenting with an inflamed mammary gland, which was diagnosed as IMC. The cell line was established from a tissue biopsy. Surface antigen marker (CD24 and CD44) expression was determined. Cystine/glutamate antiporter (xCT) expression was determined by Western blotting, flow cytometry and fluorescence immunostaining, and sulfasalazine was administered to ascertain if it suppressed xCT expression. Stem cell marker (Nanog, Sox2, Myc and Klf4) expression and aldehyde dehydrogenase (ALDH) activity were also investigated. The cultured cells showed xCT, and its suppression showed downregulation of stem cell markers and ALDH activity. Stable cell proliferation was verified. A new canine IMC-derived cell line was established. In the future, we aim to study the effect of xCT on the maintenance of cancer stem cell properties in canine tumours, and propose a new therapeutic method for the treatment of canine IMC by targeting xCT.

Inflammatory mammary carcinoma (IMC) is a rare disease with a poor prognosis and one affecting dogs. Inflammatory breast carcinoma (IBC) is a subtype of malignant breast cancer in humans with a high degree of malignancy and a similarly poor prognosis. Since the clinical symptoms and prognoses of both are similar, canine IMC has been considered as a model of human IBC. In this study, we newly established a stable IMC-derived cell line from a patient at the Yamaguchi University Animal Medical Center in Japan.

Enhancer of zeste homologue 2 (EZH2) is the human homologue of Drosophila zeste gene enhancer. The aim of this study was to determine the expression of EZH2 in canine mammary carcinomas (CMCs) and its relationship with clinicopathological features. The expression of EZH2 mRNA and protein in 53 CMC tissue and 8 normal mammary gland tissue samples was measured by quantitative real-time PCR and immunohistochemical staining assay, respectively. The relationship between EZH2 protein expression and clinicopathological features was analysed by χ2 test to further explore the clinical significance of EZH2 in CMCs. Compared with normal mammary gland tissues, EZH2 mRNA expressions were significantly increased in CMC tissues (P < 0.01). Moreover, normal mammary glands did not express the EZH2 protein but carcinomic glands did, and expression increased in CMCs with high histological grades, especially in histological grade II (P < 0.05). However, EZH2 expression was not related to age, tumour size, or metastasis (P > 0.05). The expression of EZH2 in one type of CMC was not significantly different from the expression in any other type (P > 0.05). EZH2 is highly expressed in CMCs, indicating that it can be used as a molecular marker for early diagnosis, prognosis, or therapy of CMCs.

Enhancer of zeste homologue 2 (EZH2) is the human homologue of Drosophila zeste gene enhancer. The aim of this study was to determine the expression of EZH2 in canine mammary carcinomas (CMCs) and its relationship with clinicopathological features.

Akabane virus (AKAV) has been detected in a variety of host species in China, but there are only limited records of its occurrence in goats. However, more attention needs to be paid to understanding the diversity of viruses in this species. The aim of the study was to explore the genotype characteristics and variation trend of AKAV and their relationship with virulence in Yunnan, China. Blood samples were collected from goats during routine surveillance of goat diseases in Yunnan province in 2019. The AKAV CX-01 strain was isolated using BHK-21 cells. To understand pathogenicity, the virus was intraperitoneally (IP) and intracerebrally (IC) inoculated into suckling mice and tissue samples were subsequently analysed histopathologically and immunohistochemically. Akabane virus CX-01 strain induced encephalitis and impairment of the central nervous system with fatal consequences. Phylogenetic analysis based on the ORF sequences of the small segments indicated that the AKAV isolate used was most closely related to the GD18134/2018 Chinese midge and bovine NM BS/1strains, while phylogenetic analysis based on the medium segments showed a close relationship between CX-01 and the Chinese GLXCH01 strain. The CX-01 isolate was related to AKAV genogroup Ia and probably originated from a recombination of different strains.

Akabane virus (AKAV) has been detected in a variety of host species in China, but there are only limited records of its occurrence in goats. However, more attention needs to be paid to understanding the diversity of viruses in this species. The aim of the study was to explore the genotype characteristics and variation trend of AKAV and their relationship with virulence in Yunnan, China.

Ochratoxin A (OTA) is a mycotoxin notably produced by Aspergillus and Penicillium spp. Bacillus subtilis fermentation extract (BSFE) contains specific enzymes which hydrolyse OTA. This study evaluated the efficiency of BSFE in ameliorating the immunotoxic and nephrotoxic effects of OTA in broiler chickens. Day-old broiler chicks were divided equally into four groups of ten: control, OTA (0.5 mg/kg feed), BSFE product (1 mL/L water) and OTA + BSFE at the same concentrations. The chicks were vaccinated against avian influenza, Newcastle disease, and infectious bronchitis, and lymphoproliferation was induced in all birds by phytohaemagglutinin-P (PHA-P). Serum samples were taken before sacrifice and organ tissue samples were taken after, in which renal function biomarkers were assayed and the presence of OTA residue was evaluated by high-performance thin-layer chromatography. Protein markers of apoptosis were determined by qPCR, and tissue lesions were examined histopathologically. Exposure to OTA significantly decreased the antibody response to the vaccines and the lymphoproliferative response to PHA-P, and significantly elevated the renal function indicators: serum urea, uric acid and creatinine. It also induced oxidative stress (reduced catalase activity and glutathione concentration), lipid peroxidation (increased malondialdehyde content), apoptosis (increased Bax and Caspase-3 and decreased Bcl-2 gene levels) and pathological lesions in kidney, bursa of Fabricius, spleen and thymus tissue. Residues of OTA were detected in the serum and tissue. BSFE mitigated most of these toxic effects. BSFE counters OTA-induced immunotoxicity and nephrotoxicity because of its content of carboxypeptidase and protease enzymes.

Ochratoxin A (OTA) is a mycotoxin notably produced by Aspergillus and Penicillium spp. Bacillus subtilis fermentation extract (BSFE) contains specific enzymes which hydrolyse OTA. This study evaluated the efficiency of BSFE in ameliorating the immunotoxic and nephrotoxic effects of OTA in broiler chickens.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography– mass spectrometry was developed. The analytical protocol used sulphuric acid extraction and solid-phase extraction purification. The developed procedure was subjected to validation in terms of linearity, selectivity, repeatability, reproducibility, limits of quantification and determination, matrix effect and uncertainty. A total of 29 honey samples were analysed for the determination of PAs and TAs. All the evaluated validation parameters fulfilled the requirements of European Commission Decision 2002/657/EC. At least one of the monitored alkaloids was determined in 52% of the samples. Among the most abundant alkaloids were echimidine, intermedine and lycopsamine. The total PA concentrations ranged from 2.2 to 147.0 μg kg⁻¹. Contrastingly, none of the monitored TAs was detected in the analysed samples. An assessment of the dietary exposure to PAs from the consumption of the contaminated honeys showed that three of them would pose a risk to consumers, especially if they were children. A sensitive method suitable for simultaneous determination of PAs and TAs in honey was developed and validated. The analysis of 29 honey samples for PAs and TAs revealed that honey destined for retail could pose a risk to consumers.

Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural contaminants of honey and respectively hepatoxic and neurotoxic compounds. Because honey is a popular constituent of the human diet, it is relevant to warrant the safety of the product. For that reason, a method for simultaneous determination of PAs and TAs in honey based on liquid chromatography– mass spectrometry was developed.