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.

Dogs with chronic kidney disease (CKD) may have alterations in the glomerular filtration barrier, including podocyte loss. Detection of podocyte mRNA in urine could be useful for assessing podocyturia in dogs with kidney disease. The objective of this study was to evaluate the presence of nephrin mRNA (NPHS1) and podocin mRNA (NPHS2) in urine sediments of dogs with naturally occurring CKD and healthy dogs. Twenty-four dogs, 14 with CKD and 10 as healthy controls, underwent clinical evaluation. The dogs with CKD were divided into two groups, according to the International Renal Interest Society criteria: stage 1 or 2 CKD (n = 5) and stage 3 or 4 CKD (n = 9). Urine was collected by catheterisation or free catch and RNA isolation from the urine sediments was optimised using glycogen as a co-precipitant. Detection of NPHS1 and NPHS2 in the sediment samples was performed using quantitative real-time PCR. Both types of mRNA were detected in samples from all groups, but the percentages of detection were higher in the group of dogs with stage 1 or 2 CKD and lower in the group of dogs with stage 3 or 4 disease. Physiological podocyturia was observed in healthy dogs, and the results suggest differential podocyturia in dogs with CKD, according to the stage of the disease, i.e. an increase in podocyturia in dogs at stage 1 or 2 and a reduction in podocyturia in dogs at stage 3 or 4.

Globally, genetically modified (GM) crops were grown on 191.7 million hectares in 2018, which were mostly sown with soybean, maize, cotton, oilseed rape, and rice. The most popular traits introduced through genetic modification include herbicide and pest insect resistance. The aim of this study was to identify and quantify genetically modified soybean used in animal feed in Poland. This research was based on the real-time PCR technique. All methods for GM soybean events were adopted from the EURL GMFF database of methods and previously verified to meet the minimum criteria of acceptance. Over 15 years of research, 665 samples were examined in total. The most common GM soybean event was MON40-3-2, tested for from the beginning of the investigation. Next, in decreasing order of frequency, were MON89788, MON87701, and A2704-12. In the majority of samples (606; 91%) GM soybeans were identified at a content level above the 0.9% GM content threshold for mandatory labelling. Only 59 soybean samples (9%) were identified as GM negative. GM negative results were mainly identified during the analyses in the last three years of the study, from 2017 to 2019. Our data clearly indicate that the majority of soybean used in Poland for animal feeding was genetically modified.

The aim of this study was to present two outbreaks of bovine abortion due to Leptospira infection in cattle herds located in the northern part of Sicily (Italy). The animals were positive for Leptospira interrogans serogroup Sejroe serovar Hardjo in a microscopic agglutination test (MAT). A total of 23 Charolaise cows (farm A) and 75 Limousine bulls and Cinisara and Modicana cows (farm B) were enrolled in this study. The blood samples were collected from all subjects at the following time points: before a cycle of intramuscular treatment with oxytetracycline dihydrate (T0), after 5–6 weeks from the treatment (T1), and every 10 weeks until seronegativisation (T2 in Farm A and T3 in Farm B). A serological test (MAT) was used for the diagnosis of leptospirosis. Two samples from farm A (2/23) and 29 samples from farm B (29/75) were positive to Leptospira interrogans, serogroup Sejroe, serovar Hardjo in the MAT. Leptospira spp. DNA was detected by real-time PCR in the urine sample of one positive cow on farm A, and in placenta and brain samples belonging to one aborted foetus on farm B. It is important to use serological and molecular diagnostic techniques complementarily to identify infected individuals.

Toll-like receptors (TLRs) play an important role in fast activation of the immune response to a variety of pathogens, including parasites. In this study, we focused on TLR2, because this receptor is one of the best known and most frequently analysed members of the TLR family. The aim of this study was to assess the effect of Trichinella spiralis on expression of TLR2 during the intestinal stage of infection. The experimental material consisted of isolates prepared from the intestines (jejunum and colon) of BALB/c mice infected with T. spiralis taken at 4, 8, and 16 days post infection. Our results based on quantitative real-time PCR showed that the mRNA level for TLR2 was statistically significantly higher in the jejuna of mice infected with T. spiralis than in this tissue of uninfected mice. In addition, the presence of TLR2 protein in the intestinal phase of trichinellosis was confirmed by a strong positive immunohistochemical reaction. Our results indicate that infection with T. spiralis changes the expression of TLR2 in the small intestine of the mouse host and suggest a contribution of these receptors to the host defence mechanisms during experimental trichinellosis.

White sturgeon iridovirus (WSIV) disease is caused by a virus of the eponymous family and is mostly triggered by stressful environmental conditions, i.e. high rearing density, excessive handling, or temporary loss of water. The aim of this study was to develop the most effective diagnostic method for quick and efficient confirmation or exclusion of the presence of WSIV. A total of 42 samples (spleen, gills, intestine, skin, kidney, and brain) were collected from eight sturgeon (Acipenser gueldenstaedtii and A. oxyrinchus) aged ≤5+ farmed or caught between 2010 and 2014 in open waters (Dąbie Lake and Szczecin Lagoon). They were tested for WSIV presence using conventional PCR, qPCR, and in situ hybridisation (ISH). In gross examination, all fish appeared to be healthy. Neither species showed clinical signs typical of WSIV infection. In the majority of cases, fragments of iridoviral DNA were found using molecular methods in the kidneys, and also in the liver, gills, and skin. The detection rate using ISH was 47.37% and most commonly the brain and kidney tissues were positive. The most efficient of the methods used was real-time PCR, with 100% effectiveness in detection of WSIV DNA. The study demonstrates the capabilities for WSIV diagnosis available to sturgeon farmers and water administrators, indicating useful methods of adequate sensitivity as well as organs to sample in order to achieve the highest probability of viral detection.

Introduction: Mycoplasma synoviae (MS) is a chicken pathogen of major economic importance. Material and Methods: Between 2010 and 2016, 906 commercial layer chicken flocks in Poland were examined for MS, and the phylogenetic relationship among the strains was established. Regionally dispersed samples were collected and tested with the use of real-time PCR to detect the 16S–23S intergenic spacer region. Positive samples were also tested with LAMP and conventional PCR to detect the vlhA gene. Results: MS genetic material was detected in 265 (29%) of the tested flocks by real-time PCR, in 227 by the LAMP method and in 202 (22%) by conventional PCR. The by-year percentage of positive samples began at 34% in 2010, rose to 44% in 2012, and declined to 29% in 2016. A phylogenetic analysis of Polish M. synoviae strains using a partial sequence of the vlhA gene showed nine genotypes (A–I), the most frequently occurring being F and C. Pathogenic Polish MS field isolates (n = 27) collected from chickens with clinical signs of infection were grouped for their characteristic symptoms: respiratory for genotypes C, E, F, and I (n = 13), EAA and a drop in laying for genotypes F, E, and C (n = 12), and synovitis for genotype A (n = 2). Conclusion: These data showed the country’s isolate diversity. The high prevalence suggests the need to introduce appropriate control programmes. This is the first report of molecular epidemiological data on M. synoviae infection in layer chickens in Poland.

Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 10⁴ CFU/mL, for lung tissue and nasal swabs it was 1.2 × 10⁵ CFU/mL, and for tonsils - 1.2 × 10⁵ CFU/mL.Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

African swine fever virus (ASFV) causes one of the most dangerous diseases of pigs and wild boar – African swine fever (ASF). Since its second introduction into Europe (in 2007), the disease has been spreading consistently, and now ASF-free European countries are at risk. Complex interactions between the host’s immune system and the virus have long prevented the development of a safe vaccine against ASF. This study analysed the possibility of neutralisation of the ASFV in vitro by sera collected from ASF-survivor animals. Two pig and three wild boar serum samples were collected from previously selected potential ASF survivors. All sera presented high antibody titres (>5 log₁₀/mL). Primary alveolar macrophages were cultured in growth medium containing 10% and 20% concentrations of selected sera and infected with a haemadsorbing ASFV strain (Pol18_28298_O111, genotype II). The progress of infection was investigated under a light microscope by observing the cytopathic effect (CPE) and the haemadsorption phenomenon. Growth kinetics were investigated using a real-time PCR assay. Haemadsorption inhibition was detected in the presence of almost all selected sera; however, the inhibition of virus replication in vitro was excluded. In all samples, a CPE and decreasing quantification cycle values of the viral DNA were found. Anti-ASFV antibodies alone are not able to inhibit virus replication. Interactions between the humoral and cellular immune response which effectively combat the disease are implicated in an ASF-survivor’s organism.

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.