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.

The prevalence of Dirofilaria repens in dogs in countries bordering Slovenia ranges from 1.5% to 47.3%. The aim of this study was to estimate its prevalence in Slovenian dogs and to present the cases of dirofilariasis diagnosed in humans from 2010 to 2020. Epidemiological data were collected and blood samples were taken from 465 dogs older than one year and born in Slovenia. A real-time PCR was performed on all samples to detect filarioid DNA, and a D. repens-and D. immitis-specific real-time PCR was performed on positive samples. Blood samples from 446 dogs were tested for Dirofilaria spp. using a modified Knott’s test. Human cases were diagnosed from histological sections of excised subcutaneous nodules. Descriptive statistics were used to characterise the samples. The one-sample nonparametric chi-squared test was used to assess whether categories of a variable were equally distributed. Three dogs’ samples tested positive for D. repens using the species-specific real-time PCR, while D. immitis DNA was not detected. The modified Knott’s test was positive in two of the three PCR-positive dogs, two of which had never travelled outside Slovenia’s borders. Four human patients with D. repens dirofilariasis were diagnosed. Since their travel history was unknown, autochthonous transmission could not be confirmed. Our study demonstrated a 0.64% prevalence of D. repens infection in dogs in Slovenia. Two cases could be autochthonous.

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.

Apocrine sweat gland carcinomas (ASGCs) are rare malignant skin tumours in dogs and humans. The literature published so far focuses mostly on the clinico-epidemiological aspect of these tumours, but little is known about their pathogenesis. In this study we aimed to determine whether the p53 gene is involved in the carcinogenesis of the apocrine sweat gland in dogs and whether ultraviolet radiation (UV) is related to it. Forty canine ASGCs were submitted to laser capture microdissection to isolate neoplastic cells, from which DNA was subsequently extracted. PCR amplification and sequencing of p53 exons 2–8 was then performed, followed by computer analysis of the obtained sequences. Sixteen mutations within the p53 gene were found in 13 tumours. The mutations involved C → T, T → C, G → A, and CC → TT transitions, C → G transversion and adenine deletion, which are gene alteration types known to be related to UV radiation in the process of skin carcinogenesis in humans. Six of the thirteen tumour cases displayed the C → T transitions in the same location in exon 4 and three of the thirteen cases displayed T → C in the same location in exon 5. The results of the present study indicate both the participation of the p53 gene and the influence of UV radiation in the formation of ASGCs in dogs.

Canine brachycephalic obstructive airway syndrome (BOAS) is a conformation-related respiratory disorder of dog breeds having congenitally flattened facial and skull anatomy. The aim of the study was to determine oxidative stress parameters, the lipid peroxidation product malondialdehyde, and antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase in BOAS patients before and after surgical treatment and in healthy brachycephalic dogs. Nine healthy brachycephalic dogs that had not undergone surgery and 39 BOAS patients were included in the study. The BOAS patients were classified as grade 1 (5/34), grade 2 (16/34), and grade 3 (13/34) based on the decrease in the radius of the airway in the larynx. In BOAS patients, oxidative stress parameters were determined before and two weeks after surgery, while in control dogs, blood samples were collected only on inclusion to the study. All BOAS patients showed various degrees of improvement in clinical signs after surgery. Significantly lower (P < 0.05) SOD activity was found in grade 2 and 3 BOAS patients than in grade 1 patients. Two weeks after surgery, a significant (P < 0.05) increase in SOD activity in grade 2 and 3 patients was observed. Antioxidant enzyme SOD may play an important role in BOAS and can be used as a biomarker of antioxidant status assessment in BOAS patients.

Dermatophytosis is a common skin disease in cats and dogs caused by Microsporum and Trichophyton fungi. Species identification and knowledge of their antifungal susceptibility are therapeutically and epidemiologically important. This study assessed the prevalence of feline and canine dermatophytosis in Iran, identified the aetiological agents molecularly and tested their antifungal susceptibility. A total of 308 companion animals (134 dogs and 174 cats) with skin lesions were examined from March 2015 to March 2018. Hair and skin samples were examined by microscopy with 20% KOH and cultured on Sabouraud dextrose agar with cycloheximide and chloramphenicol. Fungal isolates were confirmed by sequencing of the internal transcribed spacer (ITS) r-DNA region. The antifungal susceptibility of dermatophytes was tested by broth microdilution assay using standard drugs. Dermatophytes were found in 130 (42.2%) samples, 62 of them feline and 68 canine. Based on sequencing of all strains, M. canis (78.5%, P<0.05), M. gypseum (10.7%), and T. mentagrophytes (10.7%) were the dermatophytes isolated. The non-dermatophyte species Nannizziopsis vriesii was also isolated from two feline dermatomycosis cases. Dogs and cats younger than one year (61.5%) showed a statistically significantly higher prevalence of infection (P<0.05). Caspofungin produced the lowest geometric mean MIC at 0.0018 μg/mL, followed by ketoconazole, terbinafine, itraconazole, miconazole, griseofulvin, clotrimazole and fluconazole, in a 0.038–1.53 μg/mL range. This is the first molecular study to identify the causes of pet dermatophytosis in north-western Iran. ITS-PCR was shown to be a useful and reliable method for the identification of closely related species of dermatophytes in clinical and epidemiological settings. The lowest MIC of caspofungin indicated that this drug was the most potent in vitro.

The aim of the study was to demonstrate a link between uncomplicated Babesia canis infection in dogs and blood concentrations of zinc and copper and erythrocytic antioxidant defence – activities of glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). The study was based on 15 naturally occurring cases of canine babesiosis with anorexia, pyrexia, depression, pale mucous membrane, splenomegaly and dark red urine. Microscopic examination of Giemsa-stained peripheral blood smears and the results of PCR confirmed B. canis infection. Seven apparently healthy dogs brought in for either a check-up or vaccination were used for comparison. The levels of the erythrocytic antioxidant enzymes - SOD and CAT - were significantly higher in the infected dogs than in cytologically negative dogs. The levels of blood micronutrients were significantly lower in the infected dogs (0.478 μg of zinc per mL vs 1.241 μg/mL and 0.722 μg of copper per mL vs 1.392 μg/mL). Oxidative stress can be posited as one of the mechanisms leading to anaemia in dogs with babesiosis, and therefore antioxidant biomarker and copper and zinc concentrations could be used as indicators of disease severity and prognostic markers.

Five-minute heart-rate variability (HRV) measurement is a useful tool for assessing the autonomic nervous system (ANS) balance in humans, but there are no studies on healthy dogs. The aim of the study was, therefore, to provide the reference ranges in small and medium-sized breeds for short-term HRV time and frequency domain (TFD) analyses. A total of 79 healthy dogs were included in the study between 2015 and 2019. Grouping by age with the breakpoint at six years and subgrouping by reproductive status and sex was imposed. All the dogs were included after physical and cardiological examinations and blood analyses. The TFD of HRV were analysed from a five-minute-long digital ECG recording after removal of non-sinus complexes. There were no statistically significant differences in any TFD parameters between age, reproductive status or sex groups. A mild increase in all time domain parameters and the high-frequency (HF) band was observed in older dogs, and the low frequency (LF):HF ratio decreased in these dogs. In males, the time domain parameters and HF band increased slightly. The normal ranges for HRV derived from short-term ECG recording in the usual clinical environment now have proposed reference ranges. Our findings suggest that accommodation time, age, sex, or reproductive status do not influence the results derived from these recordings, indicating that this method is reliable for assessing the ANS function in small and medium-sized dog breeds.

Atrial fibrillation may potentially contribute to oxidative stress to a greater extent than chronic heart failure. The aim of the study was to compare the serum total antioxidant capacity and enzymatic antioxidant defence of dogs with chronic heart failure and atrial fibrillation with those of subjects with chronic heart failure and sinus rhythm and healthy controls. A total of 33 dogs were divided into three groups: dogs with chronic heart failure and atrial fibrillation (CHF + AF; n = 12), chronic heart failure and sinus rhythm (CHF + SR; n = 9), and healthy controls (n = 12). Serum total antioxidant capacity (TAC), serum CuZn-superoxide dismutase (SOD) and catalase, and plasma glutathione peroxidase (GPx) activity were determined. SOD activity and serum TAC were significantly lower in the study groups than in control animals. Catalase activity was significantly higher and plasma GPx activity significantly lower in dogs with CHF + AF compared with the CHF + SR and control dogs. The results suggest that chronic heart failure in dogs significantly impacts the serum TAC and the antioxidant enzymatic defence, while plasma GPx activity is markedly lower in dogs with chronic heart failure and atrial fibrillation. The role of that imbalance needs further investigation.

Introduction: Farm mink (Neovison vison) can be naturally exposed to T. canis and T. leonina pathogens on the farm. If mink were hosts, it would imply some veterinary public health as well as animal welfare issues. For this reason, the aim of the study was to determine whether mink might be definitive or paratenic hosts of these parasites. Material and Methods: Four groups of mink were infected with both parasite species using larvated eggs or feed containing mouse tissue previously infected with the parasites. Following inoculation, the infections were monitored in vivo by faecal examination for 14 weeks p.i., and then western blotting and ELISA were performed. Results: Coprology did not reveal any canine roundworm eggs, neither were nematodes found in mink intestines during post mortem examination. The specific IgG antibodies recognising excretory/secretory (ES) antigens of both parasite species were identified in mink sera. Single T. leonina tissue larvae were found in digested organs. Conclusions: Our results confirm that farm mink may contribute both T. canis and T. leonina infections. It was proved that farm mink were not their definitive hosts, and therefore mink faeces need not be considered a source of canine roundworm eggs in any soil it fertilises. Nonetheless, as farm mink may be a paratenic host for both parasite species, this may have some impact on the health and welfare of infected animals.