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.

Ornithobacterium rhinotracheale (ORT) causes significant economic losses to the poultry industry around the world. The bacterium often affects poultry as part of multiple infections causing very serious clinical signs that are usually not limited only to the respiratory system. This study’s main objective was the retrospective detection and identification of ORT in turkey flocks. ORT identification was performed in 6,225 samples taken from 133 different flocks between 2015 and 2020. Molecular methods were used, specifically real-time PCR and traditional PCR. We focused on partial 16S rRNA gene sequences of isolates, which were compared with sequences obtained from GenBank. The reaction products were analysed phylogenetically. Molecular methods indicating secondary infections was carried out, and the bacterial composition of the upper respiratory tract was 16S metasequenced for selected flocks to identify any other pathogens. The presence of ORT was detected in 30.83% of samples by real-time PCR and 28.57% by PCR. Phylogenetic analysis of the PCR products from the turkeys samples showed that their sequences resolved into two main genetic groups. Tests for the occurrence of secondary infections showed the presence of Mycoplasma gallisepticum and M. synoviae in some samples but the total absence of Bordetella avium. The upper respiratory tract in turkeys was dominated by two major phyla Firmicutes and Proteobacteria. At the genus level, the genera Ornithobacterium, Mycoplasma, Gallibacterium, Avibacterium, and Escherichia-Shigella were found which may include pathogenic bacteria that can cause clinical symptoms. The results of the analysis of multiple infection carried out in flocks with respiratory signs are probably associated with outbreaks of ornithobacteriosis in turkey flocks in Poland.

Seal parapoxvirus (SPPV) infection has been reported among pinnipeds in aquaria in Japan; however, its seroprevalence is unknown. Therefore, an enzyme-linked immunosorbent assay (ELISA) was developed for serological diagnosis of SPPV infection. The gene encoding the major envelope protein of SPPV was cloned into the eukaryotic expression vector pAcGFP1-N1, which encodes the green fluorescence protein (GFP), thereby producing a fusion protein (Env-GFP). Parental and cloned vector DNA was independently transfected into cultured seal cells for the expression of GFP and Env-GFP. The wells of an ELISA plate were coated with either GFP- or Env-GFP-transfected cell lysates. The light absorbance of each serum sample was adjusted by subtracting the absorbance of GFP-coated wells from that of Env-GFP-coated wells. Sera from two spotted seals (Phoca largha), six beluga whales (Delphinapterus leucas), three Pacific white-sided dolphins (Lagenorhynchus obliquidens), and ten bottlenose dolphins (Tursiops truncatus) from an aquarium in Japan were examined using the ELISA. Positive reactions were not observed, except in one preserved sample collected ten years ago from a naturally SPPV-infected spotted seal. The established ELISA could be useful in screening marine mammal sera for anti-SPPV antibodies.

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.

It has been suggested that coagulase-negative staphylococci can serve as reservoirs of virulence genes for other bacteria. This study assessed the presence of such genes in selected isolates recovered from meat of the giant African snail (Achatina achatina). Virulence genes were detected using a polymerase chain reaction targeting specific primers. Two representative isolates were identified using 16S rRNA gene sequencing. The results showed that the staphylococcal enterotoxin A gene (sea) was present in five out of the eight isolates studied. The isolates expressed resistance mainly to three antibiotics: chloramphenicol, norfloxacin and cloxacillin in descending order of incidence. Most importantly, the Staphylococcus sciuri isolate NEDU 181, in addition to being resistant to the three aforementioned antibiotics, also harboured the sea gene. Our findings demonstrate, for the first time, the presence of toxigenic and antibiotic-resistant coagulase-negative Staphylococcus spp. in commercially-available fresh snail meat. With staphylococcal enterotoxin A known to survive cooking temperature, this presents a food safety concern.

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.

Porcine circovirus 4 (PCV4) was first discovered in 2019 in a herd of pigs with porcine respiratory disease, dermatitis and nephropathy syndrome in Hunan Province, China. It has subsequently been detected in other provinces and in South Korea. In consideration of the potential of the virus to cause an epidemic, rapid, sensitive, and specific detection of PCV4 is needed, as is the facilitation of further epidemiological research through elucidation of the whole genome of PCV4. This study had those two aims. Fifty-five blood samples, two pig tissue samples, nine saliva swabs and one semen sample which all originated from Sichuan province pig farms were analysed. The virus’ genome of 1,770 bp was synthesised artificially based on a Chinese reference strain and primers and probes for the ORF2 gene were designed. Then, the amplified target fragment was cloned into the pMD19-T vector and a series of diluted recombinant plasmids were used to generate a standard curve. An optimised real-time TaqMan PCR method was established. The results of this study showed that the established method is specific for PCV4 but not for other viruses, and has amplification efficiency of 99.6%, a regression squared value (R²) of 1.000 and a detection limit of 2.2×10 DNA copies. This method was shown to be analytically specific and sensitive with a low intra- and inter-assay coefficient of variation (<1.67 %). Of a total of 67 clinical samples tested using the established method, three were shown to be positive (4%). This study confirms the existence of PCV4 in Sichuan and provides a promising alternative tool for rapid detection of PCV4.

The aim of the study was to present cases of botulism in animals found in Poland in 2019–2021. The analytical laboratory diagnosis and difficulties that occurred in the interpretation of the results are described. From 2019 to 2021, samples of serum, intestinal content, liver, spleen, kidney, faeces, wet feed, dry feed, ensilage, water and mixed samples of internal organs associated with 10 suspected animal botulism cases were sent to the National Veterinary Research Institute. Samples were analysed using a mouse bioassay and culture methods in combination with ntnh and bont gene detection. Among the ten putative botulism cases, only four (40%) were confirmed in the laboratory on the basis of the detection of botulinum toxin (BoNT) or the ntnh or bont genes. The remaining six (60%) were determined as probable despite observable characteristic clinical signs. The diagnosis of botulism in animals is a very difficult task, made so by the heterogeneity of Clostridium botulinum strains and possible loss of toxinogenicity during laboratory processing or the potential degradation of toxins. Laboratory diagnosis is a complex and problematic process which should utilise different prescribed methods for specific types of sample.

Staphylococcus aureus is a Gram-positive and round-shaped bacterium. It is often positive for catalase and nitrate reduction. Pathogenic isolates support infections by producing protein toxins and the expression of a cell-surface protein virulence factors. Sepsis-related to methicillin-resistant S. aureus (MRSA) has significant morbidity and high mortality rates (15-30%). The methicillin resistance for S. aureus is coded with the MecA gene, while the methicillin sensitivity is coded with the Nuc gene, and they are chromosomal. Similarly, it is coded with the coagulase gene for S. aureus (Coa). In this study, the 16S rRNA gene identification by Real-Time PCR was investigated in forty S. aureus isolates, which were cultured at different times in terms of MIC and SIR tests. The isolates used in the study were determined at the gene level in terms of their differences in methicillin resistance gene (MecA), methicillin susceptibility gene (Nuc), coagulase gene (Coa) and intraspecies differences were examined.As a result of the study, Staphylococcus spp. yielded positive results with 16S rRNA gene-specific primers in all isolates. Real-Time PCR analysis of the isolates with SYBRGreen-based PCR analysis was performed with 16S rRNA gene-specific primers, and the samples were confirmed to be Staphylococcus. Analysis at the family level was followed by Coa, Nuc, and MecA gene Real-Time PCR results, and it was found that, in terms of Coa and Nuc genes, 19 isolates were positive and 21 isolates were negative. In terms of MecA gene, 16 isolates were positive according to the positive sigmoidal curves and to the single peak melting values, whereas 24 isolates were found to be negative.It is thought that this study will benefit the community by contributing to the rapid and effective treatment and diagnosis of infections caused by coagulase-positive/negative Staphylococci.